Protection, restoration and enhancement of erythropoietin responsive cells, tissues and organs

ABSTRACT

Methods and compositions are provided for protecting or enhancing an erythropoietin-responsive cell, tissue, organ or body part function or viability in vivo, in situ or ex vivo in mammals, including human beings, by systemic or local administration of an erythropoietin receptor activity modulator, such as an erythropoietin or a modified erythropoietin.

This application claims the benefit of priority of PCT application no.PCT/US01/49479 filed Dec. 28, 2001 and provisional application No.60/259,245 filed Dec. 29, 2000 under 35 U.S.C. §119(e)(1), both of whichare incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

For many years, the only clear physiological role of erythropoietin hadbeen its control of the production of red blood cells. Recently, severallines of evidence suggest that erythropoietin, as a member of thecytokine superfamily, performs other important physiologic functionswhich are mediated through interaction with the erythropoietin receptor(erythropoietin-R). These actions include mitogenesis, modulation ofcalcium influx into smooth muscle cells and neural cells, and effects onintermediary metabolism. It is believed that erythropoietin providescompensatory responses that serve to improve hypoxic cellularmicroenvironment as well as modulate programmed cell death caused bymetabolic stress. Although studies have established that erythropoietininjected intracranially protects neurons against hypoxic neuronalinjury, intracranial administration is an impractical and unacceptableroute of administration for therapeutic use, particularly for normalindividuals. Furthermore, previous studies of anemic patients givenerythropoietin have concluded that peripherally-administerederythropoietin is not transported into the brain (Marti et al., 1997,Kidney Int. 51:416-8; Juul et al., 1999, Pediatr. Res. 46:543-547; Buemiet al., 2000, Nephrol. Dial. Transplant. 15:422-433.).

Various modified forms of erythropoietin have been described withactivities directed towards improving the erythropoietic activity of themolecule, such as those with altered amino acids at the carboxy terminusdescribed in U.S. Pat. No. 5,457,089 and in U.S. Pat. No. 4,835,260;erythropoietin isoforms with various numbers of sialic acid residues permolecule, such as described in U.S. Pat. No. 5,856,292; polypeptidesdescribed in U.S. Pat. No. 4,703,008; agonists described in U.S. Pat.No. 5,767,078; peptides which bind to the erythropoietin receptor asdescribed in U.S. Pat. Nos. 5,773,569 and 5,830,851; and small-moleculemimetics as described in U.S. Pat. No. 5,835,382.

It is towards the use of an erythropoietin for protecting, maintaining,enhancing, or restoring erythropoietin-responsive cells and associatedcells, tissues and organs in situ as well as ex vivo, and to delivery ofan erythropoietin across an endothelial cell barrier for the purpose ofprotecting and enhancing erythropoietin-responsive cells and associatedcells, tissues and organs distal to the vasculature, or to carryassociated molecules, that the present invention is directed.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to the use oferythropoietins for the preparation of pharmaceutical compositions forprotecting, maintaining, enhancing, or restoring the function orviability of erythropoietin-responsive mammalian cells and theirassociated cells, tissues and organs. In one particular aspect, theerythropoietin-responsive mammalian cells and their associated cells,tissue or organ are distal to the vasculature by virtue of a tightendothelial cell barrier. In another particular aspect, the cells,tissues, organs or other bodily parts are isolated from a mammalianbody, such as those intended for transplant. By way of non-limitingexamples, the erythropoietin-responsive cell or tissue may be neuronal,retinal, muscle, heart, lung, liver, kidney, small intestine, adrenalcortex, adrenal medulla, capillary endothelial, testes, ovary, pancreasor endometrial cells or tissue. These examples oferythropoietin-responsive cells are merely illustrative. In one aspect,the erythropoietin-responsive cell or its associated cells, tissues, ororgans are not excitable cells, tissues, or organs, or do notpredominantly comprise excitable cells or tissues. In a particularembodiment, the mammalian cell, tissue or organ for which anaforementioned erythropoietin derivative is used are those that haveexpended or will expend a period of time under at least one conditionadverse to the viability of the cell, tissue or organ. Such conditionsinclude traumatic in-situ hypoxia or metabolic dysfunction,surgically-induced in-situ hypoxia or metabolic dysfunction, or in-situtoxin exposure, the latter may be associated with chemotherapy orradiation therapy. In one embodiment, the adverse conditions are aresult of cardio-pulmonary bypass (heart-lung machine), as is used forcertain surgical procedures.

The erythropoietins are useful for the therapeutic or prophylactictreatment of human diseases of the CNS or peripheral nervous systemwhich have primarily neurological or psychiatric symptoms, as well asophthalmic diseases, cardiovascular diseases, cardiopulmonary diseases,respiratory diseases, kidney, urinary and reproductive diseases,gastrointestinal diseases and endocrine and metabolic abnormalities.

The invention is also directed to pharmaceutical compositions comprisingparticular erythropoietin derivatives for administration to a mammaliananimal, preferably a human. Such pharmaceutical compositions may beformulated for oral, intranasal, or parenteral administration, or in theform of a perfusate solution for maintaining the viability of cells,tissues or organs ex vivo.

Erythropoietin derivatives useful for the aforementioned purposes may beany native erythropoietin, or an erythropoietin analog, anerythropoietin mimetic, and erythropoietin fragment, a hybriderythropoietin molecule, an erythropoietin-receptor-binding molecule, anerythropoietin agonist, a renal erythropoietin, a brain erythropoietin,an oligomer thereof, a multimer thereof, a mutein thereof, a congenerthereof, a naturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, a glycosylation variant thereof, adeglycosylated variant thereof or a combination thereof. Any form oferythropoietin capable of benefitting erythropoietin-responsive cells isembraced in this aspect of the invention.

Other erythropoietin derivatives useful for the aforementioned purposesand pharmaceutical compositions include both native erythropoietins aswell as erythropoietins that have been altered by at least onemodification as compared to native erythropoietin, and preferably ascompared to native human erythropoietin. The at least one modificationmay be a modification of at least one amino acid of the erythropoietinmolecule, or a modification of at least one carbohydrate of theerythropoietin molecule. Of course, erythropoietin molecules useful forthe purposes herein may have a plurality of modifications compared tothe native molecule, such as multiple modifications of the amino acidportion of the molecule, multiple modifications of the carbohydrateportion of the molecule, or at least one modification of the amino acidportion of the molecule and at least one modification of thecarbohydrate portion of the molecule. The modified erythropoietinmolecule retains its ability of protecting, maintaining, enhancing orrestoring the function or viability of erythropoietin-responsivemammalian cells, yet other properties of the erythropoietin moleculeunrelated to the aforementioned, desirable feature may be absent ascompared to the native molecule. In a preferred embodiment, theerythropoietin derivative is non-erythropoietic.

In one embodiment, the erythropoietin of the invention has at least nosialic acid moieties. In a preferred embodiment, the modifiederythropoietin is asialoerythropoietin, and most preferably, humanasialoerythropoietin. In another embodiment, the modified erythropoietinhas 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acid moieties.

In a second embodiment, the modified erythropoietin has at least noN-linked or no O-linked carbohydrates.

In a third embodiment, the modified erythropoietin has at least areduced carbohydrate content by virtue of treatment of erythropoietinwith its native carbohydrates with at least one glycosidase.

In a fourth embodiment, the carbohydrate portion of the modifiederythropoietin molecule has at least a non-mammalian glycosylationpattern by virtue of the expression of a recombinant erythropoietin innon-mammalian cells. In preferred embodiments, the modifiederythropoietins are expressed in insect cells or plant cells.

In a fifth embodiment, the modified erythropoietin has at least one ormore oxidized carbohydrates which also may be chemically reduced. In apreferred embodiment, the modified erythropoietin is periodate-oxidizederythropoietin; in another preferred embodiment, the periodate-oxidizederythropoietin is chemically reduced with a borohydride salt such assodium borohydride or sodium cyanoborohydride.

In a sixth embodiment, the modified erythropoietin for theaforementioned uses has at least one or more modified arginine residues.In one embodiment, the modified erythropoietin comprises a glyoxalmoiety on the one or more arginine residues, such as an arylglyoxal oralkylglyoxal moiety. In another embodiment, at least one arginineresidue is modified by reaction with a vicinal diketone such as but notlimited to 2,3-butanedione or cyclohexanedione.

In a seventh embodiment, the modified erythropoietin comprises at leastone or more modified lysine residues or a modification of the N-terminalamino group of the erythropoietin molecule, such modifications as thoseresulting from reaction of the lysine residue or N-terminal amino groupwith an amino-group-modifying agent. The modified lysine residue furthermay be chemically reduced. In one preferred embodiment, anerythropoietin is biotinylated or carbamylated via one or more lysinegroups. In another preferred embodiment, the lysine is reacted with analdehyde or reducing sugar to form an imine, which may be stabilized byreduction as with sodium cyanoborohydride to form an N-alkylated lysinesuch as glucitolyl lysine, or which in the case of reducing sugars maybe stabilized by Amadori or Heyns rearrangement to form an alpha-deoxyalpha-amino sugar such as alpha-deoxy-alpha-fructosyllysine. In anotherpreferred embodiment, the lysine group is carbamylated (carbamoylated),such as by virtue of reaction with cyanate ion, alkyl-carbamylated,aryl-carbamylated, or aryl-thiocarbamylated with an alkyl-isocyanate,aryl-isocyanate, or aryl-isothiocyanate, respectively, or it may beacylated by a reactive alkylcarboxylic or arylcarboxylic acidderivative, such as by reaction with acetic anhydride, succinicanhydride or phthalic anhydride. At least one lysine group may also betrinitrophenyl modified by reaction with a trinitrobenzenesulfonic acid,or preferably its salts. In another embodiment, lysine residues may bemodified by reaction with a glyoxal derivative, such as reaction withglyoxal; methylglyoxal or 3-deoxyglucosone to form the correspondingalpha-carboxyalkyl derivatives.

In an eighth embodiment, at least one tyrosine residue of erythropoietinmay be modified in an aromatic ring position by an electrophilicreagent, such as by nitration or iodination.

In ninth embodiment, at least an aspartic acid or a glutamic acidresidue of an erythropoietin may be modified, such as by reaction with acarbodiimide followed by reaction with an amine such as but not limitedto glycinamide.

In a tenth embodiment, at least a tryptophan residue of anerythropoietin is modified, such as by reaction with n-bromosuccinimideor n-chlorosuccinimide.

In an eleventh embodiment, a modified erythropoietin molecule isprovided having at least one erythropoietin amino group removed, such asby reaction with ninhydrin followed by reduction of the resultingcarbonyl group by reaction with borohydride.

In a twelfth embodiment, a modified erythropoietin is provided having atleast an opening of at least one of the cystine linkages in theerythropoietin molecule by reaction with a reducing agent such asdithiothreitol, followed by reaction of the subsequent sulfhydryls withiodoacetamide, iodoacetic acid or another electrophile to preventreformation of the disulfide linkages.

In a thirteenth embodiment, a modified erythropoietin is provided havingat least one substitution of any one of a number of amino acids, such asa leucine, with at least one of lysine, arginine, tryptophan, tyrosine,or cysteine residues of erythropoietin, using molecular biologicaltechniques.

In a fourteenth embodiment, a modified erythropoietin is subjected to alimited chemical proteolysis that targets specific residues, forexample, to cleave after tryptophan residues. Such resultingerythropoietin fragments are embraced herein.

As noted above, an erythropoietin useful for the purposes herein mayhave at least one of the aforementioned modifications, but may have morethan one of the above modifications. By way of example of a modifiederythropoietin with one modification to the carbohydrate portion of themolecule and one modification to the amino acid portion, a modifiederythropoietin is asialoerythropoietin and has its lysine residuesbiotinylated or carbamylated. The present invention also embracescompositions, including pharmaceutical compositions, comprising one ormore of the aforementioned erythropoietins.

In another aspect of the invention, a method is provided for theprotecting, maintaining, enhancing or restoring the function orviability of erythropoietin-responsive mammalian cells and theirassociated cells, tissues and organs, by administering an effectiveamount of any one or more of the aforementioned erythropoietins. In oneparticular aspect of the method, the erythropoietin-responsive mammaliancells and their associated cells, tissue or organ are distal to thevasculature by virtue of a tight endothelial cell barrier. In anotherparticular aspect, the cells, tissues, organs or other bodily parts areisolated from a mammalian body, such as those intended for transplant.By way of non-limiting examples, the erythropoietin-responsive cell ortissue may be neuronal, retinal, muscle, heart, lung, liver, kidney,small intestine, adrenal cortex, adrenal medulla, capillary endothelial,testes, ovary, or endometrial cells or tissue. These examples oferythropoietin-responsive cells are merely illustrative. In a particularembodiment, the erythropoietin-responsive cell or its associated cells,tissues, or organs are not excitable cells, tissues, or organs, or donot predominantly comprise excitable cells or tissues. In anotherparticular embodiment, the mammalian cell, tissue or organ for which anaforementioned erythropoietin derivative may be administered are thosethat have expended or will expend a period of time under at least onecondition adverse to the viability of the cell, tissue or organ. Suchconditions may include traumatic in-situ hypoxia or metabolicdysfunction, surgically-induced in-situ hypoxia or metabolicdysfunction, or in-situ toxin exposure, the latter may be associatedwith chemotherapy or radiation therapy. In one embodiment, the inventionprotects against the adverse conditions resulting from cardio-pulmonarybypass.

In another aspect of the invention, any of the foregoing erythropoietinsas well as any other erythropoietin molecules including native humanerythropoietin can be used in the preparation of a pharmaceuticalcomposition for ex-vivo treatment of cells, tissues and organs for thepurpose of protecting, maintaining, enhancing, or restoring the functionor viability of erythropoietin-responsive mammalian cells and theirassociated cells, tissues and organs. Such ex-vivo treatment is useful,for example, for the preservation of cells, tissues or organs fortransplant, whether autotransplant or xenotransplant. The cells, tissueor organ may be bathed in a solution comprising erythropoietin, or theperfusate instilled into the organ through the vasculature or othermeans, to maintain cellular functioning during the period wherein thecells, tissue or organ is not integrated with the vasculature of thedonor or recipient. Administration of the perfusate may be made to adonor prior to organ harvesting, as well as to the harvested organ andto the recipient. Moreover, the aforementioned use of any erythropoietinis useful whenever a cell, tissue or organ is isolated from thevasculature of the individual and thus essentially existing ex vivo fora period of time, the term isolated referring to restricting or clampingthe vasculature of or to the cell, tissue, organ or bodily part, such asmay be performed during surgery, including, in particular,cardio-pulmonary bypass surgery; bypassing the vasculature of the cell,tissue, organ or bodily part; removing the cell, tissue, organ or bodilypart from the mammalian body, such may be done in advance ofxenotransplantation or prior to and during autotransplantation; ortraumatic amputation of a cell, tissue, organ or bodily part. Thus, thisaspect of the invention pertains both to the perfusion with anerythropoietin in situ and ex vivo. Ex vivo, the erythropoietin may beprovided in a cell, tissue or organ preservation solution. For eitheraspect, the exposing may be by way of continuous perfusion, pulsatileperfusion, infusion, bathing, injection, or catheterization.

In yet a further aspect, the invention is directed to a method forprotecting, maintaining, enhancing, or restoring the viability of amammalian cell, tissue, organ or bodily part which includes anerythropoietin-responsive cell or tissue, in which the cell, tissue,organ or bodily part is isolated from the mammalian body. The methodincludes at least exposing the isolated mammalian cell, tissue, organ orbodily part to an amount of an erythropoietin for a duration which iseffective to protect, maintain, enhance, or restore the aforesaidviability. In non-limiting examples, isolated refers to restricting orclamping the vasculature of or to the cell, tissue, organ or bodilypart, such as may be performed during surgery, in particular,cardio-pulmonary bypass surgery; bypassing the vasculature of the cell,tissue, organ or bodily part; removing the cell, tissue, organ or bodilypart from the mammalian body, such may be done in advance ofxenotransplantation or prior to and during autotransplantation; ortraumatic amputation of a cell, tissue, organ or bodily part. Thus, thisaspect of the invention pertains both to the perfusion with anerythropoietin in situ and ex vivo. Ex vivo, the erythropoietin may beprovided in a cell, tissue or organ preservation solution. For eitheraspect, the exposing may be by way of continuous perfusion, pulsatileperfusion, infusion, bathing, injection, or catheterization.

In the aforementioned isolation or ex-vivo embodiment, a usefulerythropoietin may be any of the aforementioned erythropoietins,including any native erythropoietin, or an erythropoietin analog, anerythropoietin mimetic, and erythropoietin fragment, a hybriderythropoietin molecule, an erythropoietin-receptor-binding molecule, anerythropoietin agonist, a renal erythropoietin, a brain erythropoietin,an oligomer thereof, a multimer thereof; a mutein thereof, a congenerthereof, a naturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, a glycosylation variant thereof, adeglycosylated variant thereof, or a combination thereof. Any form oferythropoietin capable of benefitting erythropoietin-responsive cells isembraced in this aspect of the invention. Other erythropoietins include,but are not limited to asialoerythropoietin, N-deglycosylatederythropoietin, O-deglycosylated erythropoietin, erythropoietin withreduced carbohydrate content, erythropoietin with altered glycosylationpatterns, erythropoietin with carbohydrates oxidized then reduced,arylglyoxal-modified erythropoietin, alkylglyoxal-modifiederythropoietin, 2,3-butanedione-modified erythropoietin,cyclohexanedione-modified erythropoietin, biotinylated erythropoietin,N-alkylated-lysyl-erythropoietin, glucitolyl lysine erythropoietin,alpha-deoxy-alpha-fructosyllysine-erythropoietin, carbamylatederythropoietin, acetylated erythropoietin, succinylated erythropoietin,alpha-carboxyalkyl erythropoietin, nitrated erythropoietin, iodinatederythropoietin, to name some representative yet non-limiting examplesbased on the teachings herein. A human erythropoietin is preferred;native human erythropoietin is most preferred. In another embodimenthuman asialoerythropoietin is preferred. In another embodiment humanphenylglyoxal erythropoietin is preferred.

By way of non-limiting examples, the aforementioned ex-vivoerythropoietin-responsive cell or tissue may be or comprise neuronal,retinal, muscle, heart, lung, liver, kidney, small intestine, adrenalcortex, adrenal medulla, capillary endothelial, testes, ovary, orendometrial cells or tissue. These examples of erythropoietin-responsivecells are merely illustrative.

All of the foregoing methods and uses are preferably applicable to humanbeings, but are useful as well for any mammal, such as but not limitedto companion animals, domesticated animals, livestock and zoo animals.Routes of administration of the aforementioned pharmaceuticalcompositions include oral, intravenous, intranasal, topical,intraluminal, inhalation or parenteral administration, the latterincluding intravenous, intraarterial, subcutaneous, intramuscular,intraperitoneal, submucosal or intradermal. For ex-vivo use, a perfusateor bath solution is preferred. This includes pervusing an isolatedportion of the vasculature in situ.

In yet another aspect of the invention, any of the aforementionederythropoietins are useful in preparing a pharmaceutical composition forrestoring a dysfunctional cell, tissue or organ when administered afterthe onset of the disease or condition responsible for the dysfunction.By way of non-limiting example, administration of a pharmaceuticalcomposition comprising erythropoietin restores cognitive function inanimals previously having brain trauma, even when administered longafter (e.g., three days, five days, a week, a month, or longer) thetrauma has subsided. Erythropoietins useful for such applicationsinclude any of the particular aforementioned erythropoietins or anynative erythropoietin, or an erythropoietin analog, an erythropoietinmimetic, and erythropoietin fragment, a hybrid erythropoietin molecule,an erythropoietin-receptor-binding molecule, an erythropoietin agonist,a renal erythropoietin, a brain erythropoietin, an oligomer thereof, amultimer thereof, a mutein thereof, a congener thereof, anaturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, a glycosylation variant thereof, adeglycosylated variant thereof, or a combination thereof. Any form oferythropoietin capable of benefitting erythropoietin-responsive cells isembraced in this aspect of the invention. Other erythropoietinderivatives useful for the aforementioned purposes and pharmaceuticalcompositions include both native erythropoietins as well aserythropoietins that have been altered by at least one modification ascompared to native erythropoietin, and preferably as compared to nativehuman erythropoietin. The at least one modification may be amodification of at least one amino acid of the erythropoietin molecule,or a modification of at least one carbohydrate of the erythropoietinmolecule. Of course, erythropoietin molecules useful for the purposesherein may have a plurality of modifications compared to the nativemolecule, such as multiple modifications of the amino acid portion ofthe molecule, multiple modifications of the carbohydrate portion of themolecule, or at least one modification of the amino acid portion of themolecule and at least one modification of the carbohydrate portion ofthe molecule. The modified erythropoietin molecule retains its abilityof protecting, maintaining, enhancing or restoring the function orviability of erythropoietin-responsive mammalian cells, yet otherproperties of the erythropoietin molecule unrelated to theaforementioned, desirable feature may be absent as compared to thenative molecule. A human erythropoietin is preferred; native humanerythropoietin is most preferred. In another embodiment humanasialoerythropoietin is preferred.

In yet another embodiment, the invention provides methods for the use ofthe aforementioned erythropoietin for restoring a dysfunctional cell,tissue or organ when administered after the onset of the disease orcondition responsible for the dysfunction. By way of non-limitingexample, methods for administration of a pharmaceutical compositioncomprising erythropoietin restores cognitive function in animalspreviously having brain trauma, even when administered long after (e.g.,three days, five days, a week, a month, or longer) the trauma hassubsided. Erythropoietins useful for such methods include any of theparticular aforementioned erythropoietins or any native erythropoietin,or an erythropoietin analog, an erythropoietin mimetic, anderythropoietin fragment, a hybrid erythropoietin molecule, anerythropoietin-receptor-binding molecule, an erythropoietin agonist, arenal erythropoietin, a brain erythropoietin, an oligomer thereof, amultimer thereof, a mutein thereof, a congener thereof, anaturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, a glycosylation variant thereof, adeglycosylated variant thereof, or a combination thereof. Any form oferythropoietin capable of benefitting erythropoietin-responsive cells isembraced in this aspect of the invention. Other erythropoietinderivatives useful for the aforementioned purposes and pharmaceuticalcompositions include both native erythropoietins as well aserythropoietins that have been altered by at least one modification ascompared to native erythropoietin, and preferably as compared to nativehuman erythropoietin. The at least one modification may be amodification of at least one amino acid of the erythropoietin molecule,or a modification of at least one carbohydrate of the erythropoietinmolecule. Of course, erythropoietin molecules useful for the purposesherein may have a plurality of modifications compared to the nativemolecule, such as multiple modifications of the amino acid portion ofthe molecule, multiple modifications of the carbohydrate portion of themolecule, or at least one modification of the amino acid portion of themolecule and at least one modification of the carbohydrate portion ofthe molecule. The modified erythropoietin molecule retains its abilityof protecting, maintaining, enhancing or restoring the function orviability of erythropoietin-responsive mammalian cells, yet otherproperties of the erythropoietin molecule unrelated to theaforementioned, desirable feature may be absent as compared to thenative molecule. A human erythropoietin is preferred; native humanerythropoietin is most preferred. In another embodiment humanasialoerythropoietin is preferred.

In still yet a further aspect of the present invention, methods areprovided for facilitating the transcytosis of a molecule across anendothelial cell barrier in a mammal by administration a composition ofa molecule in association with an erythropoietin such as: anerythropoietin having at least no sialic acid moieties; anerythropoietin having at least no N-linked or no O-linked carbohydrates;an erythropoietin having at least a reduced carbohydrate content byvirtue of treatment of native erythropoietin with at least oneglycosidase; an erythropoietin with a carbohydrate portion of theerythropoietin molecule haying at least a non-mammalian glycosylationpattern by virtue of the expression of a recombinant erythropoietin innon-mammalian cells; an erythropoietin has at least one or more oxidizedcarbohydrates which also may be chemically reduced; an erythropoietinhaving at least one or more modified arginine residues; anerythropoietin having at least one or more modified lysine residues or amodification of the N-terminal amino group of the erythropoietinmolecule; an erythropoietin having at least a modified tyrosine residue;an erythropoietin having at least a modified aspartic acid or a glutamicacid residue; an erythropoietin having at least a modified tryptophanresidue; an erythropoietin having at least one amino group removed; anerythropoietin having at least an opening of at least one of the cystinelinkages in the erythropoietin molecule; an erythropoietin is providedhaving at least one substitution of at least one amino acid; or atruncated erythropoietin.

The association between the molecule to be transported and theerythropoietin may be, for example, a labile covalent bond, a stablecovalent bond, or a noncovalent association with a binding site for themolecule. Endothelial cell barriers may be the blood-brain barrier, theblood-eye barrier, the blood-testes barrier, the blood-ovary barrier andthe blood-placenta barrier. Suitable molecule for transport by themethod of the present invention include hormones, such as growthhormone, antibiotics and anti-cancer agents.

It is a further aspect of the present invention to provide a compositionfor facilitating the transcytosis of a molecule across an endothelialcell barrier in a mammal, said composition comprising said molecule inassociation with an erythropoietin such as an erythropoietin having atleast no sialic acid moieties; an erythropoietin having at least noN-linked or no O-linked carbohydrates; an erythropoietin having at leasta reduced carbohydrate content by virtue of treatment of nativeerythropoietin with at least one glycosidase; an erythropoietin with acarbohydrate portion of the modified erythropoietin molecule having atleast a non-mammalian glycosylation pattern by virtue of the expressionof a recombinant erythropoietin in non-mammalian cells; anerythropoietin has at least one or more oxidized carbohydrates whichalso may be chemically reduced; an erythropoietin having at least one ormore modified arginine residues; an erythropoietin having at least oneor more modified lysine residues or a modification of the N-terminalamino group of the erythropoietin molecule; an erythropoietin having atleast a modified tyrosine residue; an erythropoietin having at least amodified aspartic acid or a glutamic acid residue; an erythropoietinhaving at least a modified tryptophan residue; an erythropoietin havingat least one amino group removed; an erythropoietin having at least anopening of at least one of the cystine linkages in the erythropoietinmolecule; an erythropoietin is provided having at least one substitutionof at least one amino acid; or a truncated erythropoietin.

The association may be, for example, a labile covalent bond, a stablecovalent bond, or a noncovalent association with a binding site for themolecule. Endothelial cell barriers may be the blood-brain barrier, theblood-eye barrier, the blood-testes barrier, the blood-ovary barrier andthe blood-placenta barrier. Suitable molecule for transport by themethod of the present invention include hormones, such as growthhormone, antibiotics and anti-cancer agents.

In a still further aspect of the present invention, any of theaforementioned erythropoietin are useful in preparing a pharmaceuticalcomposition for facilitating the transcytosis of a molecule across anendothelial cell barrier in a mammal, said composition comprising saidmolecule in association with an erythropoietin such as an erythropoietinhaving at least no sialic acid moieties; an erythropoietin having atleast no N-linked or no O-linked carbohydrates; an erythropoietin havingat least a reduced carbohydrate content by virtue of treatment of nativeerythropoietin with at least one glycosidase; an erythropoietin with acarbohydrate portion of the modified erythropoietin molecule having atleast a non-mammalian glycosylation pattern by virtue of the expressionof a recombinant erythropoietin in non-mammalian cells; anerythropoietin has at least one or more oxidized carbohydrates whichalso may be chemically reduced; an erythropoietin having at least one ormore modified arginine residues; an erythropoietin having at least oneor more modified lysine residues or a modification of the N-terminalamino group of the erythropoietin molecule; an erythropoietin having atleast a modified tyrosine residue; an erythropoietin having at least amodified aspartic acid or a glutamic acid residue; an erythropoietinhaving at least a modified tryptophan residue; an erythropoietin havingat least one amino group removed; an erythropoietin having at least anopening of at least one of the cystine linkages in the erythropoietinmolecule; an erythropoietin is provided having at least one substitutionof at least one amino acid; or a truncated erythropoietin.

The association may be, for example, a labile covalent bond, a stablecovalent bond, or a noncovalent association with a binding site for themolecule. Endothelial cell barriers may be the blood-brain barrier, theblood-eye barrier, the blood-testes barrier, the blood-ovary barrier andthe blood-placenta barrier. Suitable molecule for transport by themethod of the present invention include hormones, such as growthhormone, antibiotics and anti-cancer agents.

These and other aspects of the present invention will be betterappreciated by reference to the following Figures and DetailedDescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the translocation of parenterally-administerederythropoietin into the cerebrospinal fluid.

FIG. 2 shows the protection of the myocardium from ischemic damage byerythropoietin after temporary vascular occlusion.

FIG. 3 shows the maintenance of the function of a heart prepared fortransplantation by erythropoietin.

FIG. 4 compares the in-vitro efficacy of erythropoietin andasialoerythropoietin on the viability of serum-starved P19 cells.

FIG. 5 is another experiment which compares the in-vitro efficacy oferythropoietin and asialoerythropoietin on the viability ofserum-starved P19 cells.

FIG. 6 compares the in-vitro efficacy of erythropoietin andphenylglyoxal-modified erythropoietin on the viability of serum-starvedP19 cells.

FIG. 7 shows protection of erythropoietin and asialoerythropoietin in arat focal cerebral ischemia model.

FIG. 8 shows a dose response comparing the efficacy of humanerythropoietin and human asialoerythropoietin in middle cerebral arteryocclusion in a model of ischemic stroke

FIG. 9 shows the effect of biotinylated erythropoietin andasialoerythropoietin in the P19 assay.

FIG. 10 shows the activity of iodinated erythropoietin in the P19 assay.

FIG. 11 depicts the effects of erythropoietin treatment in a ratglaucoma model.

FIG. 12 shows the extent of preservation of retinal function byerythropoietin in the rat glaucoma model.

FIG. 13 depicts the restoration of cognitive function following braintrauma by administration of erythropoietin starting five days aftertrauma.

FIG. 14 depicts the restoration of cognitive function following braintrauma by administration of erythropoietin starting 30 days aftertrauma.

FIG. 15 depicts the efficacy of human asialoerythropoietin in a kainatemodel of cerebral toxicity.

DETAILED DESCRIPTION OF THE INVENTION

“Erythropoietin-responsive cell” refers to a mammalian cell whosefunction or viability may be maintained, promoted, enhanced,regenerated, or in any other way benefitted, by exposure to anerythropoietin. Non-limiting examples of such cells include neuronal,retinal, muscle, heart, lung, liver, kidney, small intestine, adrenalcortex, adrenal medulla, capillary endothelial, testes, ovary, andendometrial cells. Moreover, such erythropoietin-responsive cells andthe benefits provided thereto by an erythropoietin may be extended toprovide protection or enhancement indirectly to other cells that are notdirectly erythropoietin responsive, or of tissues or organs whichcontain such non-erythropoietin-responsive cells. These other cells, ortissues or organs which benefit indirectly from the enhancement oferythropoietin-responsive cells present as part of the cells, tissue ororgan as “associated” cells, tissues and organs. Thus, benefits of anerythropoietin as described herein may be provided as a result of thepresence of a small number or proportion of erythropoietin-responsivecells in a tissue or organ, for example, excitable or neuronal tissuepresent in such tissue, or the Leydig cells of the testis, which makestestosterone. In one aspect, the erythropoietin-responsive cell or itsassociated cells, tissues, or organs are not excitable cells, tissues,or organs, or do not predominantly comprise excitable cells or tissues.

The methods of the invention provide for the local or systemicprotection or enhancement of cells, tissues and organs within amammalian body, under a wide variety of normal and adverse conditions,or protection of those which are destined for relocation to anothermammalian body. In addition, restoration or regeneration of dysfunctionis also provided. As mentioned above, the ability of an erythropoietinto cross a tight endothelial cell barrier and exert its positive effectson erythropoietin-responsive cells (as well as other types of cells)distal to the vasculature offers the potential to prevent as well astreat a wide variety of conditions and diseases which otherwise causesignificant cellular and tissue damage in an animal, including human,and moreover, permit success of heretofore unattemptable surgicalprocedures for which risk traditionally outweighed the benefits. Theduration and degree of purposeful adverse conditions induced forultimate benefit, such as high-dose chemotherapy, radiation therapy,prolonged ex-vivo transplant survival, and prolonged periods ofsurgically-induced ischemia, may be carried out by taking advantage ofthe invention herein. However, the invention is not so limited, butincludes as one aspect, methods or compositions wherein the targeterythropoietin-responsive cells are distal to the vasculature by virtueof an endothelial-cell barrier or endothelial tight junctions. Ingeneral, the invention is directed to any erythropoietin-responsivecells and associated cells, tissues and organs which may benefit fromexposure to an erythropoietin. Furthermore, cellular, tissue or organdysfunction may be restored or regenerated after an acute adverse event(such as trauma) by exposure to an erythropoietin.

The invention is therefore directed generally to the use oferythropoietin for the preparation of pharmaceutical compositions forthe aforementioned purposes in which cellular function is maintained,promoted, enhanced, regenerated, or in any other way benefitted. Theinvention is also directed to methods for maintaining, enhancing,promoting, or regenerating cellular function by administering to amammal an effective amount of an erythropoietin as described herein. Theinvention is further directed to methods for maintaining, promoting,enhancing, or regenerating cellular function ex vivo by exposing cells,a tissue or organ to an erythropoietin. The invention is also directedto a perfusate composition comprising an erythropoietin for use in organor tissue preservation.

The various methods of the invention utilize a pharmaceuticalcomposition which at least includes an erythropoietin at an effectiveamount for the particular route and duration of exposure to exertpositive effects or benefits on erythropoietin-responsive cells withinor removed from a mammalian body. Where the target cell, tissues ororgans of the intended therapy require the erythropoietin to cross anendothelial cell barrier, the pharmaceutical composition includes theerythropoietin at a concentration which is capable, after crossing theendothelial cell barrier, of exerting its desirable effects upon theerythropoietin-responsive cells. Molecules capable of interacting withthe erythropoietin receptor and modulating the activity of the receptor,herein referred to as erythropoietin or erythropoietin receptor activitymodulators, are useful in the context of the present invention. Thesemolecules may be, for example, naturally-occurring, synthetic, orrecombinant forms of erythropoietin molecules, as described above, orother molecules which may not necessarily resemble erythropoietin in anymanner, except to modulate erythropoietin responsive cell activity, asdescribed herein.

Erythropoietin is a glycoprotein hormone which in humans has a molecularweight of about 34 kDa. The mature protein comprises 165 amino acids,and the glycosyl residues comprise about 40% of the weight of themolecule. The forms of erythropoietin useful in the practice of thepresent invention encompass naturally-occurring, synthetic andrecombinant forms of the following human and other mammalianerythropoietin-related molecules: erythropoietin, asialoerythropoietin,deglycosylated erythropoietin, erythropoietin analogs, erythropoietinmimetics, erythropoietin fragments, hybrid erythropoietin molecules,erythropoietin receptor-binding molecules, erythropoietin agonists,renal erythropoietin, brain erythropoietin, oligomers and multimersthereof, muteins thereof, and congeners thereof. In addition,erythropoietin forms useful in the practice of the present inventioninclude proteins that represent functionally equivalent gene products.Such an equivalent erythropoietin gene product include mutanterythropoietins, which may contain deletions, including internaldeletions, additions, including additions yielding fusion proteins, orconservative substitutions of amino acid residues within and/or adjacentto the amino acid sequence, but that result in a “silent” change, inthat the change produces a functionally equivalent erythropoietin. Suchamino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Alternatively,non-conservative amino acid changes, and larger insertions and deletionsmay be used to create functionally altered erythropoietin mutants. Suchmutants can be used to alter erythropoietin properties in desirableways. For example, in one embodiment, an erythropoietin useful for thepractice of the invention can be a mutant erythropoietin altered in oneor more amino acids within the four functional domains of erythropoietinwhich affect receptor binding: VLQRY and/or TKVNFYAW and/or SGLRSLTTLand/or SNFLRG. In another embodiment, erythropoietins containingmutations in the surrounding areas of the molecule which affect thekinetics or receptor-binding properties of the molecule can be used.

The term “erythropoietin” as well as “an erythropoietin” may be usedinterchangeably or conjunctively, and the various analogs, fragments,hybrid molecules, agonists, muteins, and other forms as described aboveembrace the variants in the extents of and sites of glycosylation oferythropoietin, including native, deglycosylated, asialylated, and otherpartially glycosylated forms of erythropoietin. Non-limiting examples ofsuch variants are described in Tsuda et al., 1990, Eur. J. Biochem.188:405-411, incorporated herein by reference. Bacteria, yeast, insect,plant, mammallian, including human. In addition, a variety of hostsystems may be used for expression and production of recombinanterythropoietin, including, but not limited to, bacteria, yeast, insect,plant, and mammalian, including human, cell systems. For example,recombinant erythropoietin produced in bacteria, which do notglycosylate or sialate the product, could be used to producenon-glycosylated forms of erythropoietin. Alternatively, recombinanterythropoietin can produced in other systems that do glycosylate, e.g.,plants, including human cells.

As noted above, the invention herein embraces any and all erythropoietinreceptor activity modulator molecules capable of exerting positiveactivity on erythropoietin-responsive cells, regardless of anystructural relationship of the molecule with erythropoietin.

In addition, erythropoietin itself may be modified to tailor itsactivities for a specific tissue or tissues. Several non-limitingstrategies which may be carried out to achieve this desired tissuespecificity include modifications that shorten circulating half-life andthus reducing the time erythropoietin can interact with erythroidprecursors, or modification of the primary structure of theerythropoietin molecule. One approach to reducing circulating half lifeis to remove or modify the glycosylation moieties, of whicherythropoietin has three N-linked and one O-linked. Such variants ofglycosylated erythropoietin can be produced in a number of ways. Forexample, the sialic acids which terminate the end of the sugar chainscan be removed by specific sialidases depending on the chemical linkageconnecting the sialic acid to the sugar chain. Alternatively, theglycosylated structure can be dismantled in different ways by usingother enzymes that cleave at specific linkages. Techniques to modify theprimary structure are myriad and include substitution of specific aminoacids, chemical modification of amino acids, or addition of otherstructures which interfere with the interaction of erythropoietin withany of its receptors. Use of such forms of erythropoietin are fullyembraced herein. In a preferred embodiment, the half-life of thenon-erythropoietic erythropoietin of the invention is reduced by about90% from that of native erythropoietin.

Some of these molecules will nevertheless mimic the actions oferythropoietin itself in other tissues or organs. For example, a 17-mercontaining the amino-acid sequence of 31-47 of native erythropoietin isinactive for erythropoiesis but fully active for neural cells in vitro(Campana & O'Brien, 1998: Int. J. Mol. Med. 1:235-41).

Furthermore, derivative erythropoietin molecules desirable for the usesdescribed herein may be generated by guanidination, amidination,carbamylation (carbamoylation), trinitrophenylation, acetylation,succinylation, nitration, or modification of arginine, lysine, tyrosine,tryptophan, or cysteine residues or carboxyl groups, among otherprocedures, such as limited proteolysis, removal of amino groups, and/ormutational substitution of arginine, lysine, tyrosine, tryptophan, orcysteine residues by molecular biological techniques to produceerythropoietin which maintain an adequate level of activities forspecific organs and tissues but not for others, such as erythrocytes(e.g., Satake et al; 1990, Biochim. Biophys. Acta 1038:125-9;incorporated herein by reference in its entirety). One non-limitingexample as described hereinbelow is the modification of erythropoietinarginine residues by reaction with a glyoxal such as phenylglyoxal(according to the protocol of Takahashi, 1977, J. Biochem. 81:395-402).As will be seen below, such an erythropoietin molecule fully retains itsneurotrophic effect. Such erythropoietin molecules are fully embracedfor the various uses and compositions described herein.

Synthetic and recombinant molecules, such as brain erythropoietin andrenal erythropoietin, recombinant mammalian forms of erythropoietin, aswell as its naturally-occurring, tumor-derived, and recombinantisoforms, such as recombinantly-expressed molecules and those preparedby homologous recombination are provided herein. Furthermore, thepresent invention includes molecules including peptides which bind theerythropoietin receptor, as well as recombinant constructs or othermolecules which possess part or all of the structural and/or biologicalproperties of erythropoietin, including fragments and multimers oferythropoietin or its fragments. Erythropoietin herein embracesmolecules with altered erythropoietin receptor binding activities,preferably with increased receptor affinity, in particular as pertainsto enhancing transport across endothelial cell barriers. Muteinscomprising molecules which have additional or reduced numbers ofglycosylation sites are included herein. As noted above, the terms“erythropoietin” and “mimetics” as well as the other terms are usedinterchangeably herein to refer to the erythropoietin-responsive cellprotective and enhancing molecules related to erythropoietin as well asthe molecules which are capable of crossing endothelial cell barriers.

Furthermore, molecules produced by transgenic animals are alsoencompassed here. It should be noted that erythropoietin molecules asembraced herein do not necessarily resemble erythropoietin structurallyor in any other manner, except for ability to interact with theerythropoietin receptor or modulate erythropoietin receptor activity oractivate erythropoietin-activated signaling cascades, as describedherein.

By way of non-limiting examples, forms of erythropoietin useful for thepractice of the present invention include erythropoietin muteins, suchas those with altered amino acids at the carboxy terminus described inU.S. Pat. No. 5,457,089 and in U.S. Pat. No. 4,835,260;asialoerythropoietin and erythropoietin isoforms with various numbers ofsialic acid residues per molecule, such as described in U.S. Pat. No.5,856,298; polypeptides described in U.S. Pat. No. 4,703,008; agonistsdescribed in U.S. Pat. No. 5,767,078; peptides which bind to theerythropoietin receptor as described in U.S. Pat. Nos. 5,773,569 and5,830,851; small-molecule mimetics which activate the erythropoietinreceptor, as described in U.S. Pat. No. 5,835,382; and erythropoietinanalogs described in WO 9505465, WO 9718318, and WO 9818926. All of theaforementioned citations are incorporated herein to the extent that suchdisclosures refer to the various alternate forms or processes forpreparing such forms of the erythropoietins of the present invention.

Erythropoietin can be obtained commercially, for example, under thetrademarks of PROCRIT, available from Ortho Biotech Inc., Raritan, N.J.,and EPOGEN, available from Amgen, Inc., Thousand Oaks, Calif.

The activity (in units) of erythropoietin (erythropoietin) anderythropoietin-like molecules is traditionally defined based on itseffectiveness in stimulating red cell production in rodent models (andas derived by international standards of erythropoietin). One unit (U)of regular erythropoietin (MW of ˜34,000) is ˜8 ng of protein (1 mgprotein is approximately 125,000 U). However, as the effect onerythropoiesis is incidental to the desired activities herein and maynot necessarily be a detectable property of certain of theerythropoietins of the invention, the definition of activity based onerythropoietic activity is inappropriate. Thus, as used herein, theactivity unit of erythropoietin or erythropoietin-related molecules isdefined as the amount of protein required to elicit the same activity inneural or other erythropoietin-responsive cellular systems as iselicited by WHO international standard erythropoietin in the samesystem. The skilled artisan will readily determine the units of anon-erythropoietic erythropoietin or related molecule following theguidance herein.

Further to the above-mentioned erythropoietin modifications usefulherein, the following discussion expands on the various erythropoietinsof the invention.

An erythropoietin of the invention may have at least no sialic acidmoieties, referred to as asialoerythropoietin. Preferably, anerythropoietin of the invention is human asialoerythropoietin. Inalternative embodiments, the erythropoietin of the invention may have atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acid residues.It may be prepared by desialylating erythropoietin using a sialidase,such as is described in the manufacturer's packaging for Sialydase Afrom ProZyme Inc., San Leandro, Calif. Typically, PROZYME® GLYCOPRO®sequencing-grade SIALYDASE A™ (N-acetylneuraminate glycohydrolase, EC3.2.1.18) is used to cleave all non-reducing terminal sialic acidresidues from complex carbohydrates and glycoproteins such aserythropoietin. It will also cleave branched sialic acids (linked to aninternal residue). Sialydase A is isolated from a clone of Arthrobacterureafaciens.

An erythropoietin may have at least a reduced number of N-linkedcarbohydrates. To remove N-linked carbohydrates, erythropoietin may betreated with hydrazine, in accordance, for example, with the methodsdescribed by Hermentin et al., 1996, Glycobiology 6(2):217-30. As notedabove, erythropoietin has three N-linked carbohydrate moieties; thepresent invention embraces those erythropoietins with two, one, or noN-linked carbohydrate.

An erythropoietin of the invention may have at least a reducedcarbohydrate content by virtue of treatment of native erythropoietinwith at least one glycosidase. For example, the procedure of Chen andEvangelista, 1998, Electrophoresis 19(15):2639-44, may be followed.Furthermore, removal of the O-linked carbohydrate may be achievedfollowing the methods described in Hokke et al., 1995, Eur. J. Biochem.228(3):981-1008.

The carbohydrate portion of an erythropoietin molecule may have at leasta non-mammalian glycosylation pattern by virtue of the expression of arecombinant erythropoietin in non-mammalian cells. Preferably, theerythropoietins are expressed in insect or plant cells. By way ofnon-limiting example, expression of erythropoietin in insect cells usinga baculovirus expression system may be carried out in accordance withQuelle et al., 1989, Blood 74(2):652-657. Another method is described inU.S. Pat. No. 5,637,477. Expression in a plant system may be carried outin accordance with the method of Matsumoto et al., 1993, Biosci.Biotech. Biochem. 57(8):1249-1252. Alternatively, expression in bacteriawill result in non-glycosylated forms of erythropoietin. These aremerely exemplary of methods useful for the production of anerythropoietin of the invention are in no way limiting.

An erythropoietin of the invention may have at least one or moreoxidized carbohydrates that also may be chemically reduced. For example,the erythropoietin may be periodate-oxidized erythropoietin; theperiodate-oxidized erythropoietin also may be chemically reduced with aborohydride salt such as sodium borohydride or sodium cyanoborohydride.Periodate oxidation of erythropoietin may be carried out, for example,by the methods described by Linsley et al., 1994, Anal. Biochem.219(2):207-17. Chemical reduction following periodate oxidation may becarried out following the methods of Tonelli and Meints, 1978, J.Supramol. Struct. 8(1):67-78.

An erythropoietin for the aforementioned uses may have at least one ormore modified arginine residues. For example, the modifiederythropoietin may comprise a R-glyoxal moiety on the one or morearginine residues, where R may be an aryl, heteroaryl, lower alkyl,lower alkoxy, or cycloalkyl group, or an alpha-deoxyglycitolyl group. Asused herein, the term lower “alkyl” means a straight- or branched-chainsaturated aliphatic hydrocarbon group preferably containing 1-6 carbonatoms. Representative of such groups are methyl, ethyl, isopropyl,isobutyl, butyl, pentyl, hexyl and the like. The term “alkoxy” means alower alkyl group as defined above attached to the remainder of themolecule by oxygen. Examples of alkoxy include methoxy, ethoxy, propoxy,isopropoxy and the like. The term “cycloalkyl” refers to cyclic alkylgroups with three up to about 8 carbons, including for examplecyclopropyl, cyclobutyl, cyclohexyl and the like. The term aryl refersto phenyl and naphthyl groups. The term heteroaryl refers toheterocyclic groups containing 4-10 ring members and 1-3 heteroatomsselected from the group consisting of oxygen, nitrogen and sulfur.Examples include but are not limited to isoxazolyl, phenylisoxazolyl,furyl, pyrimidinyl, quinolyl, tetrahydroquinolyl, pyridyl, imidazolyl,pyrrolidinyl, 1,2,4-triazoylyl, thiazolyl, thienyl, and the like. The Rgroup may be substituted, as for example the 2,3,4-trihydroxybutyl groupof 3-deoxyglucosone. Typical examples of R-glyoxal compounds areglyoxal, methylglyoxal, 3-deoxyglucosone, and phenylglyoxal. PreferredR-glyoxal compounds are methylglyoxal or phenylglyoxal. An exemplarymethod for such modification may be found in Werber et al., 1975, Isr.J. Med. Sci. 11(11): 1169-70, using phenylglyoxal.

In a further example, at least one arginine residue may be modified byreaction with a vicinal diketone such as 2,3-butanedione orcyclohexanedione, preferably in ca. 50 millimolar borate buffer at pH8-9. A procedure for the latter modification with 2,3-butanedione may becarried out in accordance with Riordan, 1973, Biochemistry 12(20):3915-3923; and that with cyclohexanone according to Patthy et al., 1975,J. Biol. Chem. 250(2): 565-9.

An erythropoietin of the invention may comprise at least one or moremodified lysine residues or a modification of the N-terminal amino groupof the erythropoietin molecule, such modifications as those resultingfrom reaction of the lysine residue with an amino-group-modifying agent.In another embodiment, lysine residues may be modified by reaction withglyoxal derivatives, such as reaction with glyoxal, methylglyoxal and3-deoxyglucosone to form alpha-carboxyalkyl derivatives. Examples arereaction with glyoxal to form carboxymethyllysine as in Glomb andMonnier, 1995, J. Biol. Chem. 270(17):10017-26, Or with methylglyoxal toform (1-carboxyethyl)lysine as in Degenhardt et al., 1998, Cell. Mol.Biol. (Noisy-le-grand) 44(7):1139-45. The modified lysine residuefurther may be chemically reduced. For example, the erythropoietin maybe biotinylated via lysine groups, such as in accordance with the methoddescribed in Example 5, in which D-biotinoyl-ε-aminocaproicacid-N-hydroxysuccinimide ester was reacted with erythropoietin,followed by removal of unreacted biotin by gel filtration on a Centricon10 column, as described by Wojchowski and Caslake, 1989, Blood74(3):952-8. In this paper, the authors use three different methods ofbiotinylating erythropoietin, any of which may be used for thepreparation of the erythropoietins for the uses herein. Biotin may beadded to (1) the sialic acid moieties (2) carboxylate groups or (3)amino groups.

In another preferred embodiment, the lysine may be reacted with analdehyde or reducing sugar to form an imine, which may be stabilized byreduction as with sodium cyanoborohydride to form an N-alkylated lysinesuch as glucitolyl lysine, or which in the case of reducing sugars maybe stabilized by Amadori or Heyns rearrangement to form an alpha-deoxyalpha-amino sugar such as alpha-deoxy-alpha-fructosyllysine. As anexample, preparation of a fructosyllysine-modified protein by incubationwith 0.5 M glucose in sodium phosphate buffer at pH 7.4 for 60 days isdescribed by Makita et al., 1992, J. Biol. Chem. 267:5133-5138. Inanother example, the lysine group may be carbamylated, such as by virtueof reaction with cyanate ion, or alkyl- or aryl-carbamylated or-thiocarbamylated with an alkyl- or aryl-isocyanate or -isothiocyanate,or it may be acylated by a reactive alkyl- or arylcarboxylic acidderivative, such as by reaction with acetic anhydride or succinicanhydride or phthalic anhydride. Exemplary are the modification oflysine groups with 4-sulfophenylisothiocyanate or with acetic anhydride,both as described in Gao et al., 1994, Proc Natl Acad Sci USA91(25):12027-30. Lysine groups may also be trinitrophenyl modified byreaction with trinitrobenzenesulfonic acid or preferably its salts. Suchmethods are described below in Example 5.

At least one tyrosine residue of an erythropoietin may be modified in anaromatic ring position by an electrophilic reagent, such as by nitrationor iodination. By way of non-limiting example, erythropoietin may bereacted with tetranitromethane (Nestler et al., 1985, J. Biol. Chem.260(12):7316-21; or iodinated as described in Example 5.

At least an aspartic acid or a glutamic acid residue of anerythropoietin may be modified, such as by reaction with a carbodiimidefollowed by reaction with an amine such as but not limited toglycinamide. Examples of such modifications may be found in Example 5.

In another example, a tryptophan residue of an erythropoietin may bemodified, such as by reaction with n-bromosuccinimide orn-chlorosuccinimide, following methods such as described in Josse etal., Chem Biol Interact 1999 May 14; 119-120.

In yet another example, an erythropoietin molecule may be prepared byremoving at least one amino group, such may be achieved by reaction withninhydrin followed by reduction of the subsequent carbonyl group byreaction with borohydride.

In still a further example, an erythropoietin is provided that has atleast an opening of at least one of the cystine linkages in theerythropoietin molecule by reaction with a reducing agent such asdithiothreitol, followed by reaction of the subsequent sulfhydryls withiodoacetamide, iodoacetic acid or another electrophile to preventreformation of the disulfide linkages.

An erythropoietin is provided having at least one substitution of anyone of a number of amino acids, such as a leucine, with at least one oflysine, arginine, tryptophan, tyrosine, or cysteine residues oferythropoietin, using molecular biological techniques.

A modified erythropoietin may be prepared by subjecting anerythropoietin to a limited chemical proteolysis that targets specificresidues, for example, to cleave after tryptophan residues. Suchresulting erythropoietin fragments are embraced herein.

As noted above, an erythropoietin useful for the purposes herein mayhave at least one of the aforementioned modifications, but may have morethan one of the above modifications. By way of example of a modifiederythropoietin with one modification to the carbohydrate portion of themolecule and one modification to the amino acid portion, a modifiederythropoietin may be asialoerythropoietin and have its lysine residuesbiotinylated or carbamylated.

Thus, various erythropoietin molecules and pharmaceutical compositionscontaining them for the uses described herein are embraced. Sucherythropoietin molecules include but are not limited toasialoerythropoietin, N-deglycosylated erythropoietin, O-deglycosylatederythropoietin, erythropoietin with reduced carbohydrate content,erythropoietin with altered glycosylation patterns, erythropoietin withcarbohydrates oxidized then reduced, arylglyoxal-modifiederythropoietin, alkylglyoxal-modified erythropoietin,2,3-butanedione-modified erythropoietin, cyclohexanedione-modifiederythropoietin, biotinylated erythropoietin,N-alkylated-lysyl-erythropoietin, glucitolyl lysine erythropoietin,alpha-deoxy-alpha-fructosyllysine-erythropoietin, carbamylatederythropoietin, acetylated erythropoietin, succinylated erythropoietin,alpha-carboxyalkyl erythropoietin, nitrated erythropoietin, iodinatederythropoietin, to name some representative yet non-limiting examplesbased on the teachings herein. Preferred are the aforementioned modifiedforms based on human erythropoietin.

Moreover, certain of the aforementioned erythropoietins are new, and theinvention is directed to such compounds as well as pharmaceuticalcompositions comprising them. By way of non-limiting example, such newerythropoietins include periodate-oxidized erythropoietin, glucitolyllysine erythropoietin, fructosyl lysine erythropoietin, 3-deoxyglucosoneerythropoietin, and carbamylated asialoerythropoietin.

A variety of host-expression vector systems may be utilized to producethe erythropoietins and erythropoietin-related molecules of theinvention. Such host-expression systems represent vehicles by which theerythropoietins of interest may be produced and subsequently purified,but also represent cells that may, when transformed or transfected withthe appropriate nucleotide coding sequences, exhibit the modifiederythropoietin gene product in situ. These include but are not limitedto, bacteria, insect, plant, mammallian, including human host systems,such as, but not limited to, insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing themodified erythropoietin product coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingerythropoietin-related molecule coding sequences; or mammalian cellsystems, including human cell systems, (e.g., HT1080, COS, CHO, BHK,293, 3T3) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells, including human host cells, include but are not limited toHT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theerythropoietin-related molecule gene product may be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci that in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell linesthat express the erythropoietin-related molecule gene product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of theerythropoietin-related molecule gene product.

Alternatively, the expression characteristic of an endogenouserythropoietin gene within a cell line or microorganism may be modifiedby inserting a heterologous DNA regulatory element into the genome of astable cell line or cloned microorganism such that the insertedregulatory element is operatively linked with the endogenouserythropoietin gene. For example, an endogenous erythropoietin genewhich is normally “transcriptionally silent”, i.e., an erythropoietingene which is normally not expressed, or is expressed only a very lowlevels in a cell line, may be activated by inserting a regulatoryelement which is capable of promoting the expression of a normallyexpressed gene product in that cell line or microorganism.Alternatively, a transcriptionally silent, endogenous erythropoietingene may be activated by insertion of a promiscuous regulatory elementthat works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such it is operatively linked with anendogenous erythropoietin gene, using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described e.g., in French Patent No. 2646438 to InstitutPasteur, U.S. Pat. No. 4,215,051 to Chappel; U.S. Pat. No. 5,578,461 toSherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

In one embodiment of the invention, an erythropoietin-related moleculedeficient in sialic residues, or completely lacking sialic residues, maybe produced in mammalian cell, including a human cell. Such cells may beengineered to be deficient in, or lacking, the enzymes that add sialicacids, i.e., the β-galactoside α2,3 sialyltransferase (“α2,3sialyltransferase”) and the β-galactoside α2,6 sialyltransferase (“α2,6sialyltransferase”) activity. In one embodiment, a mammalian cell isused in which either or both the α2,3 sialyltransferase gene and/or theα2,6 sialyltransferase gene, is deleted. Such deletions may beconstructed using gene knock-out techniques well known in the art. Inanother embodiment, dihydrofolate reductase (DHFR) deficient ChineseHamster Ovary (CHO) cells are used as the host cell for the productionof recombinant erythropoietin-related molecules. CHO cells do notexpress the enzyme α2,6 sialyltransferase and therefore do not addsialic acid in the 2,6 linkage to N-linked oligosaccharides ofglycoproteins produced in these cells. As a result, recombinant proteinsproduced in CHO cells lack sialic acid in the 2,6 linkage to galactose(Sasaki et al. (1987; Takeuchi et al. supra; Mutsaers et al Eur. J.Biochem. 156, 651 (1986); Takeuchi et al. J. Chromotgr. 400, 207 (1987).In one embodiment, to produce a host cell for the production ofasialo-erythropoietin, the gene encoding α2,3 sialyltransferase in CHOcells is deleted. Such a 2,3 sialyltransferase knock-out CHO cellscompletely lack sialyltransferase activity, and as a result, are usefulfor the recombinant expression and production of asialo-erythropoietin.

In another embodiment, asialo glycoproteins can be produced byinterfering with sialic acid transport into the golgi apparatus e.g.,Eckhardt et al., 1998, J. Biol. Chem. 273:20189-95). Using methods wellknown to those skilled in the art (e.g., Oelmann et al., 2001, J. Biol.Chem. 276:26291-300), mutagenesis of the nucleotide sugar CMP-sialicacid transporter can be accomplished to produce mutants of Chinesehamster ovary cells. These cells cannot add sialic acid residues toglycoproteins such as erthropoietin and produce onlyasialoerythropoietin. Transfected mammalian cells producingerythropoietin also produce cytosolic sialidase which if it leaks intothe culture medium degrades sialoerythropoietin with high efficiency(e.g., Gramer et al, 1995 Biotechnology 13:692-698). Using methods wellknown to those knowledgeable in the art (e.g., from information providedin Ferrari et al, 1994, Glycobiology 4:367-373), cell lines can betransfected, mutated or otherwise caused to constitutively producesialidase. In this manner, asialoerythropoietin can be produced duringthe manufacture of asialoerythropoietin.

In the practice of one aspect of the present invention, a pharmaceuticalcomposition as described above containing an erythropoietin may beadministerable to a mammal by any route which provides a sufficientlevel of an erythropoietin in the vasculature to permit translocationacross an endothelial cell barrier and beneficial effects onerythropoietin-responsive cells. When used for the purpose of perfusinga tissue or organ, similar results are desired. In the instance whereinthe erythropoietin is used for ex-vivo perfusion, the erythropoietin maybe any form of erythropoietin, such as the aforementionederythropoietins but not limited thereto an may be inclusive of nativeerythropoietins including human erythropoietin. In the instance wherethe cells or tissue is non-vascularized and/or the administration is bybathing the cells or tissue with the composition of the invention, thepharmaceutical composition provides an effectiveerythropoietin-responsive-cell-beneficial amount of an erythropoietin.The endothelial cell barriers across which an erythropoietin maytranslocate include tight junctions, perforated junctions, fenestratedjunctions, and any other types of endothelial barriers present in amammal. A preferred barrier is an endothelial cell tight junction, butthe invention is not so limiting.

The aforementioned erythropoietins are useful generally for thetherapeutic or prophylactic treatment of human diseases of the centralnervous system or peripheral nervous system which have primarilyneurological or psychiatric symptoms, ophthalmic diseases,cardiovascular diseases, cardiopulmonary diseases, respiratory diseases,kidney, urinary and reproductive diseases, gastrointestinal diseases andendocrine and metabolic abnormalities. In particular, such conditionsand diseases include hypoxic conditions, which adversely affectexcitable tissues, such as excitable tissues in the central nervoussystem tissue, peripheral nervous system tissue, or cardiac tissue orretinal tissue such as, for example, brain, heart, or retina/eye.Therefore, the invention can be used to treat or prevent damage toexcitable tissue resulting from hypoxic conditions in a variety ofconditions and circumstances. Non-limiting examples of such conditionsand circumstances are provided in the table hereinbelow.

In the example of the protection of neuronal tissue pathologiestreatable in accordance with the present invention, such pathologiesinclude those which result from reduced oxygenation of neuronal tissues.Any condition which reduces the availability of oxygen to neuronaltissue, resulting in stress, damage, and finally, neuronal cell death,can be treated by the methods of the present invention. Generallyreferred to as hypoxia and/or ischemia, these conditions arise from orinclude, but are not limited to stroke, vascular occlusion, prenatal orpostnatal oxygen deprivation, suffocation, choking, near drowning,carbon monoxide poisoning, smoke inhalation, trauma, including surgeryand radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructivepulmonary disease, emphysema, adult respiratory distress syndrome,hypotensive shock, septic shock, anaphylactic shock, insulin shock,sickle cell crisis, cardiac arrest, dysrhythmia, nitrogen narcosis, andneurological deficits caused by heart-lung bypass procedures.

In one embodiment, for example, the specific EPO compositions can beadministered to prevent injury or tissue damage resulting from risk ofinjury or tissue damage during surgical procedures, such as, forexample, tumor resection or aneurysm repair. Other pathologies caused byor resulting from hypoglycemia which are treatable by the methodsdescribed herein include insulin overdose, also referred to asiatrogenic hyperinsulinemia, insulinoma, growth hormone deficiency,hypocortisolism, drug overdose, and certain tumors.

Other pathologies resulting from excitable neuronal tissue damageinclude seizure disorders, such as epilepsy, convulsions, or chronicseizure disorders. Other treatable conditions and diseases includediseases such as stroke, multiple sclerosis, hypotension, cardiacarrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brainor spinal cord trauma, AIDS dementia, age-related loss of cognitivefunction, memory loss, amyotrophic lateral sclerosis, seizure disorders,alcoholism, retinal ischemia, optic nerve damage resulting fromglaucoma, and neuronal loss.

The specific compositions and methods of the invention may be used totreat conditions of, and damage to, retinal tissue. Such disordersinclude, but are not limited to retinal ischemia, macular degeneration,retinal detachment, retinitis pigmentosa, arteriosclerotic retinopathy,hypertensive retinopathy, retinal artery blockage, retinal veinblockage, hypotension, and diabetic retinopathy.

In another embodiment, the methods principles of the invention may beused to protect or treat injury resulting from radiation damage toexcitable tissue. A further utility of the methods of the presentinvention is in the treatment of neurotoxin poisoning, such as domoicacid shellfish poisoning, neurolathyrism, and Guam disease, amyotrophiclateral sclerosis, and Parkinson's disease.

As mentioned above, the present invention is also directed to a methodfor enhancing excitable tissue function in a mammal by peripheraladministration of an erythropoietin as described above. Various diseasesand conditions are amenable to treatment using this method, and further,this method is useful for enhancing cognitive function in the absence ofany condition or disease. These uses of the present invention aredescribe in further detail below and include enhancement of learning andtraining in both human and non-human mammals.

Conditions and diseases treatable by the methods of this aspect of thepresent invention directed to the central nervous system include but arenot limited to mood disorders, anxiety disorders, depression, autism,attention deficit hyperactivity disorder, and cognitive dysfunction.These conditions benefit from enhancement of neuronal function. Otherdisorders treatable in accordance with the teachings of the presentinvention include sleep disruption, for example, sleep apnea andtravel-related disorders; subarachnoid and aneurismal bleeds,hypotensive shock, concussive injury, septic shock, anaphylactic shock,and sequelae of various encephalitides and Meningitides, for example,connective tissue disease-related cerebritides such as lupus. Other usesinclude prevention of or protection from poisoning by neurotoxins, suchas domoic acid shellfish poisoning, neurolathyrism, and Guam disease,amyotrophic lateral sclerosis, Parkinson's disease; postoperativetreatment for embolic or ischemic injury; whole brain irradiation;sickle cell crisis; and eclampsia.

A further group of conditions treatable by the methods of the presentinvention include mitochondrial dysfunction, of either an hereditary oracquired nature, which are the cause of a variety of neurologicaldiseases typified by neuronal injury and death. For example, Leighdisease (subacute necrotizing encephalopathy) is characterized byprogressive visual loss and encephalopathy, due to neuronal drop out,and myopathy. In these cases, defective mitochondrial metabolism failsto supply enough high energy substrates to fuel the metabolism ofexcitable cells. An erythropoietin receptor activity modulator optimizesfailing function in a variety of mitochondrial diseases. As mentionedabove, hypoxic conditions adversely affect excitable tissues. Theexcitable tissues include, but are not limited to, central nervoussystem tissue, peripheral nervous system tissue, and heart tissue. Inaddition to the conditions described above, the methods of the presentinvention are useful in the treatment of inhalation poisoning such ascarbon monoxide and smoke inhalation, severe asthma, adult respiratorydistress syndrome, and choking and near drowning. Further conditionswhich create hypoxic conditions or by other means induce excitabletissue damage include hypoglycemia that may occur in inappropriatedosing of insulin, or with insulin-producing neoplasms (insulinoma).

Various neuropsychologic disorders which are believed to originate fromexcitable tissue damage are treatable by the instant methods. Chronicdisorders in which neuronal damage is involved and for which treatmentby the present invention is provided include disorders relating to thecentral nervous system and/or peripheral nervous system includingage-related loss of cognitive function and senile dementia, chronicseizure disorders, Alzheimer's disease, Parkinson's disease, dementia,memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberoussclerosis, Wilson's Disease cerebral and progressive supranuclear palsy,Guam disease, Lewy body dementia, prion diseases, such as spongiformencephalopathies, e.g., Creutzfeldt-Jakob disease, Huntington's disease,myotonic dystrophy, Freidrich's ataxia and other ataxias, as well asGilles de la Tourette's syndrome, seizure disorders such as epilepsy andchronic seizure disorder, stroke, brain or spinal cord trauma, AIDSdementia, alcoholism, autism, retinal ischemia, glaucoma, autonomicfunction disorders such as hypertension and sleep disorders, andneuropsychiatric disorders that include, but are not limited toschizophrenia, schizoaffective disorder, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, panic disorder, as well as unipolar and bipolar affectivedisorders. Additional neuropsychiatric and neurodegenerative disordersinclude, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which in incorporated herein byreference in its entirety.

In another embodiment, recombinant chimeric toxin molecules comprisingerythropoietin can be used for therapeutic delivery of toxins to treat aproliferative disorder, such as cancer, or viral disorder, such assubacute sclerosing panencephalitis.

The following table lists additional exemplary, non-limiting indicationsas to the various conditions and diseases amenable to treatment by theaforementioned erythropoietins.

Cell, tissue or Dysfunction or organ pathology Condition or disease TypeHeart Ischemia Coronary artery Acute, chronic disease Stable, unstableMyocardial Dressler's syndrome infarction Angina Congenital heartValvular disease Cardiomyopathy Prinzmetal angina Cardiac ruptureAneurysmatic Septal perforation Angiitis Arrhythmia Tachy-, Stable,unstable bradyarrhythmia Hypersensitive carotid sinus Supraventricular,node ventricular Conduction abnormalities Congestive heart Left, right,bi- Cardiomyopathies, such as failure ventricular idiopathic familial,infective, metabolic, storage disease, deficiencies, connective tissuedisorder, infiltration and granulomas, neurovascular MyocarditisAutoimmune, infective, idiopathic Cor pulmonale Blunt and penetratingtrauma Toxins Cocaine Vascular Hypertension Primary, secondaryDecompression sickness Fibromuscular hyperplasia Aneurysm Dissecting,ruptured, enlarging Lungs Obstructive Asthma Chronic bronchitis,Emphysema and airway obstruction Ischemic lung disease Pulmonaryembolism, Pulmonary thrombosis, Fat embolism Environmental lung diseasesIschemic lung disease Pulmonary embolism Pulmonary thrombosisInterstitial lung Idiopathic pulmonary disease fibrosis CongenitalCystic fibrosis Cor pulmonale Trauma Pneumonia and Infectious,parasitic, pneumonitides toxic, traumatic, burn, aspiration SarcoidosisPancreas Endocrine Diabetes mellitus, Beta cell failure, dysfunctiontype I and II Diabetic neuropathy Other endocrine cell failure of thepancreas Exocrine Exocrine pancreas pancreatitis failure Bone OsteopeniaPrimary Hypogonadism secondary immobilisation Postmenopausal Age-relatedHyperparathyroidism Hyperthyroidism Calcium, magnesium, phosphorusand/or vitamin D deficiency Osteomyelitis Avascular necrosis TraumaPaget's disease Skin Alopecia Areata Primary Totalis Secondary Malepattern baldness Vitiligo Localized Primary generalized secondaryDiabetic ulceration Peripheral vascular disease Burn injuries AutoimmuneLupus disorders erythematodes, Sjiogren, Rheumatoid arthritis,Glomerulonephritis, Angiitis Langerhan's histiocytosis Eye Opticneuritis Blunt and penetrating injuries, Infections, Sarcoid, Sickle Cdisease, Retinal detachment, Temporal arteritis Embryonic and Asphyxiafetal disorders Ischemia CNS Chronic fatigue syndrome, acute and chronichypoosmolar and hyperosmolar syndromes, AIDS Dementia, ElectrocutionEncephalitis Rabies, Herpes Meningitis Subdural hematoma Nicotineaddiction Drug abuse and Cocaine, heroin, withdrawal crack, marijuana,LSD, PCP, poly-drug abuse, ecstasy, opioids, sedative hypnotics,amphetamines, caffeine Obsessive- compulsive disorders Spinal stenosis,Transverse myelitis, Guillian Barre, Trauma, Nerve root compression,Tumoral compression, Heat stroke ENT Tinnitus Meuniere's syndromeHearing loss Traumatic injury, barotrauma Kidney Renal failure Acute,chronic Vascular/ischemic, interstitial disease, diabetic kidneydisease, nephrotic syndromes, infections Henoch S. Purpura Striatedmuscle Autoimmune Myasthenia gravis disorders DermatomyositisPolymyositis Myopathies Inherited metabolic, endocrine and toxic Heatstroke Crush injury Rhabdomylosis Mitochondrial disease InfectionNecrotizing fasciitis Sexual Central and Impotence secondary dysfunctionperipheral to medication Liver hepatitis Viral, bacterial, parasiticIschemic disease Cirrhosis, fatty liver Infiltrative/metabolic diseasesGastrointestinal Ischemic bowel disease Inflammatory bowel diseaseNecrotizing enterocolitis Organ Treatment of donor transplantation andrecipient Reproductive tract infertility Vascular Autoimmune Uterineabnormalities Implantation disorders Endocrine Glandular hyper- andhypofunction

As mentioned above, these diseases, disorders or conditions are merelyillustrative of the range of benefits provided by the erythropoietins ofthe invention. Accordingly, this invention generally providestherapeutic or prophylactic treatment of the consequences of mechanicaltrauma or of human diseases. Therapeutic or prophylactic treatment fordiseases, disorders or conditions of the CNS and/or peripheral nervoussystem are preferred. Therapeutic or prophylactic treatment fordiseases, disorders or conditions which have a psychiatric component isprovided. Therapeutic or prophylactic treatment for diseases, disordersor conditions including but not limited to those having an ophthalmic,cardiovascular, cardiopulmonary, respiratory; kidney, urinary,reproductive, gastrointestinal, endocrine, or metabolic component isprovided.

In one embodiment, such a pharmaceutical composition of anerythropoietin may be administered systemically to protect or enhancethe target cells, tissue or organ. Such administration may beparenterally, via inhalation, or transmucosally, e.g., orally, nasally,rectally, intravaginally, sublingually, submucosally or transdermally.Preferably, administration is parenteral, e.g., via intravenous orintraperitoneal injection, and also including, but is not limited to,intra-arterial, intramuscular, intradermal and subcutaneousadministration.

For other routes of administration, such as by use of a perfusate,injection into an organ, or other local administration, a pharmaceuticalcomposition will be provided which results in similar levels of anerythropoietin as described above. A level of about 15 pM-30 nM ispreferred.

The pharmaceutical compositions of the invention may comprise atherapeutically effective amount of a compound, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized foreign pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions in water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. The compounds ofthe invention can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for oral administration may beprovided as capsules or tablets; as powders or granules; as solutions,syrups or suspensions (in aqueous or non-aqueous liquids); as ediblefoams or whips; or as emulsions. Tablets or hard gelatine capsules maycomprise lactose, starch or derivatives thereof, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, stearic acid or saltsthereof. Soft gelatine capsules may comprise vegetable oils, waxes,fats, semi-solid, or liquid polyols etc. Solutions and syrups maycomprise water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract (e.g., glycerylmonostearate or glyceryl distearate may be used). Thus, the sustainedrelease of an active agent may be achieved over many hours and, ifnecessary, the active agent can be protected from being degraded withinthe stomach. Pharmaceutical compositions for oral administration may beformulated to facilitate release of an active agent at a particulargastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.Pharmaceutical compositions adapted for topical administration may beprovided as ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. For topical administration tothe skin, mouth, eye or other external tissues a topical ointment orcream is preferably used. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops. In these compositions, the active ingredient can be dissolved orsuspended in a suitable carrier, e.g., in an aqueous solvent.Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions adapted for nasal and pulmonaryadministration may comprise solid carriers such as powders (preferablyhaving a particle size in the range of 20 to 500 microns). Powders canbe administered in the manner in which snuff is taken, i.e., by rapidinhalation through the nose from a container of powder held close to thenose. Alternatively, compositions adopted for nasal administration maycomprise liquid carriers, e.g., nasal sprays or nasal drops.Alternatively, inhalation directly into the lungs may be accomplished byinhalation deeply or installation through a mouthpiece into theoropharynx. These compositions may comprise aqueous or oil solutions ofthe active ingredient. Compositions for administration by inhalation maybe supplied in specially adapted devices including, but not limited to,pressurized aerosols, nebulizers or insufflators, which can beconstructed so as to provide predetermined dosages of the activeingredient. In a preferred embodiment, pharmaceutical compositions ofthe invention are administered into the nasal cavity directly or intothe lungs via the nasal cavity or oropharynx.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which may contain antioxidants, buffers, bacteriostats andsolutes that render the compositions substantially isotonic with theblood of an intended recipient. Other components that may be present insuch compositions include water, alcohols, polyols, glycerine andvegetable oils, for example. Compositions adapted for parenteraladministration may be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, e.g., sterile saline solution for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.In one embodiment, an autoinjector comprising an injectable solution ofan erythropoietin may be provided for emergency use by ambulances,emergency rooms, and battlefield situations, and even forself-administration in a domestic setting, particularly where thepossibility of traumatic amputation may occur, such as by imprudent useof a lawn mower. The likelihood that cells and tissues in a severed footor toe will survive after reattachment may be increased by administeringan erythropoietin to multiple sites in the severed part as soon aspracticable, even before the arrival of medical personnel on site, orarrival of the afflicted individual with severed toe in tow at theemergency room.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically-sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile saline can be providedso that the ingredients may be mixed prior to administration.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

A perfusate composition may be provided for use in transplanted organbaths, for in situ perfusion, or for administration to the vasculatureof an organ donor prior to organ harvesting. Such pharmaceuticalcompositions may comprise levels of an erythropoietin or a form of anerythropoietin not suitable for acute or chronic, local or systemicadministration to an individual, but will serve the functions intendedherein in a cadaver, organ bath, organ perfusate, or in situ perfusateprior to removing or reducing the levels of the erythropoietin containedtherein before exposing or returning the treated organ or tissue toregular circulation. The erythropoietin for this aspect of the inventionmay be any erythropoietin, such as naturally-occurring forms such ashuman erythropoietin, or any of the erythropoietin hereinabovedescribed, such as asialoerythropoietin andphenylglyoxal-erythropoietins, as non-limiting examples.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In another embodiment, for example, erythropoietin can be delivered in acontrolled-release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRCCrit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507;Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment,the compound can be delivered in a vesicle, in particular a liposome(see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes inthe Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); WO 91/04014; U.S. Pat. No.4,704,355; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). Inanother embodiment, polymeric materials can be used [see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Press:Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley: New York (1984);Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1953;see also Levy et al., 1985, Science 228:190; During et al., 1989, AnnNeurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the target cells, tissue ororgan, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, pp. 115-138 in Medical Applications of Controlled Release, vol.2, supra, 1984). Other controlled release systems are discussed in thereview by Langer (1990, Science 249:1527-1533).

In another embodiment, erythropoietin, as properly formulated, can beadministered by nasal, oral, rectal, vaginal, or sublingualadministration.

In a specific embodiment, it may be desirable to administer theerythropoietin compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as silastic membranes, or fibers.

Selection of the preferred effective dose will be determined by askilled artisan based upon considering several factors which will beknown to one of ordinary skill in the art. Such factors include theparticular form of erythropoietin, and its pharmacokinetic parameterssuch as bioavailability, metabolism, half-life, etc., which will havebeen established during the usual development procedures typicallyemployed in obtaining regulatory approval for a pharmaceutical compound.Further factors in considering the dose include the condition or diseaseto be treated or the benefit to be achieved in a normal individual, thebody mass of the patient, the route of administration, whetheradministration is acute or chronic, concomitant medications, and otherfactors well known to affect the efficacy of administered pharmaceuticalagents. Thus the precise dosage should be decided according to thejudgment of the practitioner and each patient's circumstances, e.g.,depending upon the condition and the immune status of the individualpatient, according to standard clinical techniques.

In another aspect of the invention, a perfusate or perfusion solution isprovided for perfusion and storage of organs for transplant, theperfusion solution including an amount of an erythropoietin effective toprotect erythropoietin-responsive cells and associated cells, tissues ororgans. Transplant includes but is not limited to xenotransplantation,where a organ (including cells, tissue or other bodily part) isharvested from one donor and transplanted into a different recipient;and autotransplant, where the organ is taken from one part of a body andreplaced at another, including bench surgical procedures, in which anorgan may be removed, and while ex vivo, resected, repaired, orotherwise manipulated, such as for tumor removal, and then returned tothe original location. In one embodiment, the perfusion solution is theUniversity of Wisconsin (UW) solution (U.S. Pat. No. 4,798,824) whichcontains from about 1 to about 25 U/ml erythropoietin, 5% hydroxyethylstarch (having a molecular weight of from about 200,000 to about 300,000and substantially free of ethylene glycol, ethylene chlorohydrin, sodiumchloride and acetone); 25 mM KH₂PO₄, 3 mM glutathione; 5 mM adenosine;10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium gluconate; 1.5 mMCaCl₂, 105 mM sodium gluconate; 200,000 units penicillin; 40 unitsinsulin; 16 mg Dexamethasone; 12 mg Phenol Red; and has a pH of 7.4-7.5and an osmolality of about 320 mOSm/l. The solution is used to maintaincadaveric kidneys and pancreases prior to transplant. Using thesolution, preservation can be extended beyond the 30-hour limitrecommended for cadaveric kidney preservation. This particular perfusateis merely illustrative of a number of such solutions that can be adaptedfor the present use by inclusion of an effective amount of anerythropoietin. In a further embodiment, the perfusate solution containsfrom about 5 to about 35 U/ml erythropoietin, or from about 10 to about30 U/ml erythropoietin. As mentioned above, any form of erythropoietincan be used in this aspect of the invention.

While the preferred recipient of an erythropoietin for the purposesherein throughout is a human, the methods herein apply equally to othermammals, particularly domesticated animals, livestock, companion and zooanimals. However, the invention is not so limiting and the benefits canbe applied to any mammal.

In further aspects of the ex-vivo invention, any erythropoietin such asbut not limited to the erythropoietins described above, as well asnative erythropoietins as well as an analog thereof, an erythropoietinmimetic, and erythropoietin fragment, a hybrid erythropoietin molecule,an erythropoietin-receptor-binding molecule, an erythropoietin agonist,a renal erythropoietin, a brain erythropoietin, an oligomer thereof, amultimer thereof, a mutein thereof, a congener thereof, anaturally-occurring form thereof, a synthetic form thereof, arecombinant form thereof, a glycosylation variant thereof, adeglycosylated variant thereof, or a combination thereof.

In another aspect of the invention, methods and compositions forenhancing the viability of cells, tissues or organs which are notisolated from the vasculature by an endothelial cell barrier areprovided by exposing the cells, tissue or organs directly to apharmaceutical composition comprising an erythropoietin, oradministering or contacting an erythropoietin-containing pharmaceuticalcomposition to the vasculature of the tissue or organ. Enhanced activityof erythropoietin-responsive cells in the treated tissue or organ areresponsible for the positive effects exerted.

As described above, the invention is based, in part, on the discoverythat erythropoietin molecules can be transported from the luminalsurface to the basement membrane surface of endothelial cells of thecapillaries of organs with endothelial cell tight junctions, including,for example, the brain, retina, and testis. Thus,erythropoietin-responsive cells across the barrier are susceptibletargets for the beneficial effects of erythropoietin, and others celltypes or tissues or organs that contain and depend in whole or in parton erythropoietin-responsive cells therein are targets for the methodsof the invention. While not wishing to be bound by any particulartheory, after transcytosis of erythropoietin, erythropoietin caninteract with an erythropoietin receptor on an erythropoietin-responsivecell, for example, neuronal, retinal, muscle, heart, lung, liver,kidney, small intestine, adrenal cortex, adrenal medulla, capillaryendothelial, testes, ovary, or endometrial cell, and receptor bindingcan initiate a signal transduction cascade resulting in the activationof a gene expression program within the erythropoietin-responsive cellor tissue, resulting in the protection of the cell or tissue, or organ,from damage, such as by toxins, chemotherapeutic agents, radiationtherapy, hypoxia, etc. Thus, methods for protectingerythropoietin-responsive cell-containing tissue from injury or hypoxicstress, and enhancing the function of such tissue are described indetail hereinbelow.

In the practice of one embodiment of the invention, a mammalian patientis undergoing systemic chemotherapy for cancer treatment, includingradiation therapy, which commonly has adverse effects such as nerve,lung, heart, ovarian or testicular damage. Administration of apharmaceutical composition comprising an erythropoietin as describedabove is performed prior to and during chemotherapy and/or radiationtherapy, to protect various tissues and organs from damage by thechemotherapeutic agent, such as to protect the testes. Treatment may becontinued until circulating levels of the chemotherapeutic agent havefallen below a level of potential danger to the mammalian body.

In the practice of another embodiment of the invention, various organswere planned to be harvested from a victim of an automobile accident fortransplant into a number of recipients, some of which required transportfor an extended distance and period of time. Prior to organ harvesting,the victim was infused with a pharmaceutical composition comprising anerythropoietin as described herein. Harvested organs for shipment wereperfused with a perfusate containing erythropoietin as described herein,and stored in a bath comprising erythropoietin. Certain organs werecontinuously perfused with a pulsatile perfusion device, utilizing aperfusate containing an erythropoietin in accordance with the presentinvention. Minimal deterioration of organ function occurred during thetransport and upon implant and reperfusion of the organs in situ.

In another embodiment of the invention, a surgical procedure to repair aheart valve required temporary cardioplegia and arterial occlusion.Prior to surgery, the patient was infused with 500 U erythropoietin perkg body weight. Such treatment prevented hypoxic ischemic cellulardamage, particularly after reperfusion.

In another embodiment of the invention, in any surgical procedure, suchas in cardiopulmonary bypass surgery, a naturally-occurringerythropoietin or any erythropoietin of the invention can be used. Inone embodiment, administration of a pharmaceutical compositioncomprising an erythropoietin as described above is performed prior to,during, and/or following the bypass procedure, to protect the functionof brain, heart, and other organs.

In the foregoing examples in which an erythropoietin of the invention,including naturally-occurring erythropoietin, is used for ex-vivoapplications, or to treat erythropoietin-responsive cells such asneuronal tissue, retinal tissue, heart, lung, liver, kidney, smallintestine, adrenal cortex, adrenal medulla, capillary endothelial,testes, ovary, or endometrial cells or tissue, the invention provides apharmaceutical composition in dosage unit form adapted for protection orenhancement of erythropoietin-responsive cells, tissues or organs distalto the vasculature which comprises, per dosage unit, an effectivenon-toxic amount within the range from about 50,000 to 500,000 Units,60,000 to 500,000 Units, 70,000 to 500,000 Units, 80,000 to 500,000Units, 90,000 to 500,000 Units, 100,000 to 500,000 Units, 150,000 to500,000 Units, 200,000 to 500,000 Units, 250,000 to 500,000 Units,300,000 to 500,000 Units, 350,000 to 500,000 Units, 400,000 to 500,000Units, or 450,000 to 500,000 Units of erythropoietin, an erythropoietinreceptor activity modulator, or an erythropoietin-activated receptormodulator and a pharmaceutically acceptable carrier. In a preferredembodiment, the effective non-toxic amount of erythropoietin is withinthe range from about 50,000 to 500,000 Units. In a preferred embodiment,the erythropoietin in the aforementioned composition isnon-erythropoietic.

In a further aspect of the invention, erythropoietin administration wasfound to restore cognitive function in animals having undergone braintrauma. After a delay of either 5 days or 30 days, administration oferythropoietin was still able to restore function as compared tosham-treated animals, indicating the ability of an erythropoietin toregenerate or restore brain activity. Thus, the invention is alsodirected to the use of an erythropoietin for the preparation of apharmaceutical composition for the treatment of brain trauma and othercognitive dysfunctions, including treatment well after the injury (e.g.three days, five days, a week, a month, or longer). The invention isalso directed to a method for the treatment of cognitive dysfunctionfollowing injury by administering an effective amount of anerythropoietin. Any erythropoietin as described herein may be used forthis aspect of the invention.

Furthermore, this restorative aspect of the invention is directed to theuse of any of the erythropoietins herein for the preparation of apharmaceutical composition for the restoration of cellular, tissue ororgan dysfunction, wherein treatment is initiated after, and well after,the initial insult responsible for the dysfunction. Moreover, treatmentusing erythropoietins of the invention can span the course of thedisease or condition during the acute phase as well as a chronic phase.

In the instance wherein an erythropoietin of the invention haserythropoietic activity, in a preferred embodiment, erythropoietin maybe administered systemically at a dosage between about 300 and about10,000 Units/kg body weight, preferably about 500-5,000 Units/kg-bodyweight, most preferably about 1,000 Units/kg-body weight, peradministration. This effective dose should be sufficient to achieveserum levels of erythropoietin greater than about 10,000, 15,000, or20,000 mU/ml of serum after erythropoietin administration. Such serumlevels may be achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hourspost-administration. Such dosages may be repeated as necessary. Forexample, administration may be repeated daily, as long as clinicallynecessary, or after an appropriate interval, e.g., every 1 to 12 weeks,preferably, every 1 to 3 weeks. In one embodiment, the effective amountof erythropoietin and a pharmaceutically acceptable carrier may bepackaged in a single dose vial or other container. In anotherembodiment, an erythropoietin useful for the purposes herein isnonerythropoietic, i.e., it is capable of exerting the activitiesdescribed herein but not causing an increase in hemoglobin concentrationor hematocrit. Such a non-erythropoietic form of erythropoietin ispreferred in instances wherein the methods of the present invention areintended to be provided chronically. In another embodiment, anerythropoietin is given at a dose greater than that necessary tomaximally stimulate erythropoiesis. As noted above, an erythropoietin ofthe invention does not necessarily have erythropoietic activity, andtherefore the above dosages expressed in hematopoietic units is merelyexemplary for erythropoietin that are erythropoietic; hereinabove molarequivalents for dosages are provided which are applicable to anyerythropoietin.

The present invention is further directed to a method for facilitatingthe transport of a molecule across an endothelial cell barrier in amammal by administering a composition which comprises the particularmolecule in association with an erythropoietin as described hereinabove.As described above, tight junctions between endothelial cells in certainorgans in the body create a barrier to the entry of certain molecules.For treatment of various conditions within the barriered organ, meansfor facilitating passage of pharmaceutical agents is desired. Anerythropoietin of the invention is useful as a carrier for deliveringother molecules across the blood-brain and other similar barriers. Acomposition comprising a molecule desirous of crossing the bather witherythropoietin is prepared, and peripheral administration of thecomposition results in the transcytosis of the composition across thebarrier. The association between the molecule to be transported acrossthe barrier and the erythropoietin may be a labile covalent bond, inwhich case the molecule is released from association with theerythropoietin after crossing the bather. If the desired pharmacologicalactivity of the molecule is maintained or unaffected by association witherythropoietin, such a complex can be administered.

The skilled artisan will be aware of various means for associatingmolecules with an erythropoietin of the invention and the other agentsdescribed above, by covalent, non-covalent, and other means;furthermore, evaluation of the efficacy of the composition can bereadily determined in an experimental system. Association of moleculeswith an erythropoietin may be achieved by any number of means, includinglabile, covalent binding, cross-linking, etc. Biotin/avidin interactionsmay be employed. As mentioned above, a hybrid molecule may be preparedby recombinant or synthetic means, for example, which includes both thedomain of the molecule with desired pharmacological activity and thedomain responsible for erythropoietin receptor activity modulation.

A molecule may be conjugated to an erythropoietin through apolyfunctional molecule, i.e., a polyfunctional crosslinker. As usedherein, the term “polyfunctional molecule” encompasses molecules havingone functional group that can react more than one time in succession,such as formaldehyde, as well as molecules with more than'one reactivegroup. As used herein, the term “reactive group” refers to a functionalgroup on the crosslinker that reacts with a functional group on amolecule (e.g., peptide, protein, carbohydrate, nucleic acid,particularly a hormone, antibiotic, or anti-cancer agent to be deliveredacross an endothelial cell barrier) so as to form a covalent bondbetween the cross-linker and that molecule. The term “functional group”retains its standard meaning in organic chemistry. The polyfunctionalmolecules which can be used are preferably biocompatible linkers, i.e.,they are noncarcinogenic, nontoxic, and substantially non-immunogenic invivo. Polyfunctional cross-linkers such as those known in the art anddescribed herein can be readily tested in animal models to determinetheir biocompatibility. The polyfunctional molecule is preferablybifunctional. As used herein, the term “bifunctional molecule” refers toa molecule with two reactive groups. The bifunctional molecule may beheterobifunctional or homobifunctional. A heterobifunctionalcross-linker allows for vectorial conjugation. It is particularlypreferred for the polyfunctional molecule to be sufficiently soluble inwater for the cross-linking reactions to occur in aqueous solutions suchas in aqueous solutions buffered at pH 6 to 8, and for the resultingconjugate to remain water soluble for more effective bio-distribution.Typically, the polyfunctional molecule covalently bonds with an amino ora sulfhydryl functional group. However, polyfunctional moleculesreactive with other functional groups, such as carboxylic acids orhydroxyl groups, are contemplated in the present invention.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., Science 223, 1304-1306 (1984). Homobifunctional molecules having atleast two active ester units include esters of dicarboxylic acids andN-hydroxysuccinimide. Some examples of such N-succinimidyl estersinclude disuccinimidyl suberate and dithio-bis-(succinimidylpropionate), and their soluble bis-sulfonic acid and bis-sulfonate saltssuch as their sodium and potassium salts. These homobifunctionalreagents are available from Pierce, Rockford, Ill.

The heterobifunctional molecules have at least two different reactivegroups. The reactive groups react with different functional groups,e.g., present on the erythropoietin and the molecule. These twodifferent functional groups that react with the reactive group on theheterobifunctional cross-linker are usually an amino group, e.g., theepsilon amino group of lysine; a sulfhydryl group, e.g., the thiol groupof cysteine; a carboxylic acid, e.g., the carboxylate on aspartic acid;or a hydroxyl group, e.g., the hydroxyl group on serine.

Of course, the various erythropoietin molecules of the invention may nothave suitable reactive groups available for use with certaincross-linking agent; however, one of skill in the art will be amplyaware of the choice of cross-linking agents based on the availablegroups for cross-linking in an erythropoietin of the invention.

When a reactive group of a heterobifunctional molecule forms a covalentbond with an amino group, the covalent bond will usually be an amido orimido bond. The reactive group that forms a covalent bond with an aminogroup may, for example, be an activated carboxylate group, ahalocarbonyl group, or an ester group. The preferred halocarbonyl groupis a chlorocarbonyl group. The ester groups are preferably reactiveester groups such as, for example, an N-hydroxy-succinimide ester group.

The other functional group typically is either a thiol group, a groupcapable of being converted into a thiol group, or a group that forms acovalent bond with a thiol group. The covalent bond will usually be athioether bond or a disulfide. The reactive group that forms a covalentbond with a thiol group may, for example, be a double bond that reactswith thiol groups or an activated disulfide. A reactive group containinga double bond capable of reacting with a thiol group is the maleimidogroup, although others, such as acrylonitrile, are also possible. Areactive disulfide group may, for example, be a 2-pyridyldithio group ora 5,5′-dithio-bis-(2-nitrobenzoic acid) group. Some examples ofheterobifunctional reagents containing reactive disulfide bonds includeN-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson, et al., 1978,Biochem J., 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce Chemical Co., Rockford, Ill. USA.

The need for the above-described conjugated to be reversible or labilemay be readily determined by the skilled artisan. A conjugate may betested in vitro for both the erythropoietin, and for the desirablepharmacological activity. If the conjugate retains both properties, itssuitability may then be tested in vivo. If the conjugated moleculerequires separation from the erythropoietin for activity, a labile bondor reversible association with erythropoietin will be preferable. Thelability characteristics may also be tested using standard in vitroprocedures before in vivo testing.

Additional information regarding how to make and use these as well asother polyfunctional reagents may be obtained from the followingpublications or others available in the art:

-   Carlsson, J. et al., 1978, Biochem. J. 173:723-737.-   Cumber, J. A. et al., 1985, Methods in Enzymology 112:207-224.-   Jue, R. et al., 1978, Biochem 17:5399-5405.-   Sun, T. T. et al., 1974, Biochem. 13:2334-2340.-   Blattler, W. A. et al., 1985, Biochem. 24:1517-152.-   Liu, F. T. et al., 1979, Biochem. 18:690-697.-   Youle, R. J. and Neville, D. M. Jr., 1980, Proc. Natl. Acad. Sci.    U.S.A. 77:5483-5486.-   Lerner, R. A. et al., 1981, Proc. Natl. Acad. Sci. U.S.A.    78:3403-3407.-   Jung, S. M. and Moroi, M., 1983, Biochem. Biophys. Acta 761:162.-   Caulfield, M. P. et al., 1984, Biochem. 81:7772-7776.-   Staros, J. V., 1982, Biochem. 21:3950-3955.-   Yoshitake, S. et al., 1979, Eur. J. Biochem. 101:395-399.-   Yoshitake, S. et al., 1982, J. Biochem. 92:1413-1424.-   Pilch, P. F. and Czech, M. P., 1979, J. Biol. Chem. 254:3375-3381.-   Novick, D. et al., 1987, J. Biol. Chem. 262:8483-8487.-   Lomant, A. J. and Fairbanks, G., 1976, J. Mol. Biol. 104:243-261.-   Hamada, H. and Tsuruo, T., 1987, Anal. Biochem. 160:483-488.-   Hashida, S. et al., 1984, J. Applied Biochem. 6:56-63.

Additionally, methods of cross-linking are reviewed by Means and Feeney,1990, Bioconjugate Chem. 1:2-12.

Barriers which are crossed by the above-described methods andcompositions of the present invention include but are not limited to theblood-brain barrier, the blood-eye barrier, the blood-testes barrier,the blood-ovary barrier, and the blood-uterus barrier.

Candidate molecules for transport across an endothelial cell barrierinclude, for example, hormones such as growth hormone, neurotrophicfactors, antibiotics or antifungals such as those normally excluded fromthe brain and other barriered organs, peptide radiopharmaceuticals,antisense drugs, antibodies against biologically-active agents,pharmaceuticals, and anti-cancer agents. Non-limiting examples of suchmolecules include growth hormone, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor(CNTF), basic fibroblast growth factor (bFGF), transforming growthfactor β1 (TGFβ1), transforming growth factor β2 (TGFβ2), transforminggrowth factor β3 (TGFβ3), interleukin 1, interleukin 2, interleukin 3,and interleukin 6, AZT, antibodies against tumor necrosis factor, andimmunosuppressive agents such as cyclosporin.

The present invention is also directed to a composition comprising amolecule to be transported via transcytosis across a endothelial celltight junction barrier and an erythropoietin as described above. Theinvention is further directed to the use of a conjugate between amolecule and an erythropoietin as described above for the preparation ofa pharmaceutical composition for the delivery of the molecule across abarrier as described above.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

Example 1 Erythropoietin Crosses the Blood-Cerebrospinal Fluid TightBarrier

Adult male Sprague-Dawley rats were anesthetized and administeredrecombinant human erythropoietin intraperitoneally. Cerebrospinal fluidwas sampled from the cisterna magna at 30 minute intervals up to 4 hrsand the erythropoietin concentration determined using a sensitive andspecific enzyme-linked immunoassay. As illustrated in FIG. 1, thebaseline erythropoietin concentration in CSF is 8 mU/ml. After a delayof several hours, the levels of erythropoietin measured in the CSF beginto rise and by 2.5 hours and later are significantly different from thebaseline concentration at the p<0.01 level. The peak level of about 100mU/ml is within the range known to exert protective effects in vitro(0.1 to 100 mU/ml). The time to peak occurs at about 3.5 hrs, which isdelayed significantly from the peak serum levels (less than 1 hr). Theresults of this experiment illustrate that significant levels oferythropoietin can be accomplished across a tight cellular junction bybolus parenteral administration of erythropoietin at appropriateconcentrations.

Example 2 Maintenance of Function in Heart Prepared for Transplantation

Wistar male rats weighing 300 to 330 g are given erythropoietin (5000U/kg body weight) or vehicle 24 h prior to removal of the heart for exvivo studies, done in accordance with the protocol of Delcayre et al.,1992, Amer. J. Physiol. 263:H1537-45. Animals are sacrificed withpentobarbital (0.3 mL), and intravenously heparinized (0.2 mL). Thehearts are initially allowed to equilibrate for 15 min The leftventricular balloon is then inflated to a volume that gives anend-diastolic pressure of 8 mm Hg. A left ventricular pressure-volumecurve is constructed by incremental inflation of the balloon volume by0.02 ml aliquots. Zero volume is defined as the point at which the leftventricular end-diastolic pressure is zero. On completion of thepressure-volume curve, the left ventricular balloon is deflated to setend-diastolic pressure back to 8 mmHg and the control period is pursuedfor 15 min, after check of coronary flow. Then the heart is arrestedwith 50 mL Celsior+molecule to rest at 4° C. under a pressure of 60 cmH₂0. The heart is then removed and stored 5 hours at 4° C. in plasticcontainer filled with the same solution and surrounded with crushed ice.

On completion of storage, the heart is transferred to a Langendorffapparatus. The balloon catheter is re-inserted into the left ventricleand re-inflated to the same volume as during preischemic period. Theheart is re-perfused for at least 2 hours at 37° C. The re-perfusionpressure is set at 50 cm H₂0 for 15 min of re-flow and then back to 100cm H₂0 for the 2 next hours. Pacing (320 beats per minute) isre-instituted. Isovolumetric measurements of contractile indexes anddiastolic pressure are taken in triplicate at 25, 45, 60, 120 min ofreperfusion. At this time point pressure volume curves are performed andcoronary effluent during the 45 nm reperfusion collected to measurecreatine kinase leakage. The two treatment groups are compared using anunpaired t-test, and a linear regression using the end-diastolicpressure data is used to design compliance curves. As shown in FIG. 2,significant improvement of left ventricular pressure developed occursafter treatment with erythropoietin, as well as improved volume-pressurecurve, decrease of left diastolic ventricular pressure and decrease ofcreatine kinase leakage.

Example 3 Erythropoietin Protects Myocardium from Ischemic Injury

Adult male rats given recombinant human erythropoietin (5000 U/kg bodyweight) 24 hrs previously are anesthetized and prepared for coronaryartery occlusion. An additional dose of erythropoietin is given at thestart of the procedure and the left main coronary artery occluded for 30minutes and then released. The same dose of erythropoietin is givendaily for one week after treatment. The animals are then studied forcardiac function. As FIG. 3 illustrates, animals receiving a shaminjection (saline) demonstrated a large increase in the left enddiastolic pressure, indicative of a dilated, stiff heart secondary tomyocardial infarction. In contradistinction, animals receivingerythropoietin suffered no decrement in cardiac function, compared tosham operated controls (difference significant at the p<0.01 level).

Example 4 Erythropoietin Molecules

Native erythropoietin may be modified to tailor its activities for aspecific tissue or tissues. Several non-limiting strategies that may becarried out to achieve this desired tissue specificity includemodifications that remove or modify the glycosylation moieties, of whicherythropoietin has three N-linked and one O-linked. Such variants ofglycosylated erythropoietin can be produced in a number of ways. Forexample, the sialic acids which terminate the end of the sugar chainscan be removed by specific sialidases depending on the chemical linkageconnecting the sialic acid to the sugar chain. Alternatively, theglycosylated structure can be dismantled in different ways by usingother enzymes that cleave at specific linkages. To validate theseprinciples, recombinant human erythropoietin was desialized usingSialidase A (Prozyme Inc.) according to the manufacturer's protocol.Successful chemical modification was confirmed by running the reactionproduct on an SDS polyacrylamide gel and staining the resultant bandswhich showed that the chemically-modified erythropoietin possessed anapparent molecular weight of ˜31 kD as expected, compared to unmodifiederythropoietin which was ˜0.34 kD and by measuring the sialic acidresidues remaining by chemical means to be <0.1 mole/mole oferythropoietin.

In another modification wherein the amino acid residues oferythropoietin are modified, arginine residues were modified by usingphenylglyoxal according to the protocol of Takahashi (1977, J. Biochem.81:395-402) carried out for variable lengths of time ranging from 0.5 to3 hrs at room temperature. The reaction was terminated by dialyzing thereaction mixture against water. Use of such modified forms oferythropoietin is fully embraced herein.

Asialoerythropoietin and phenylglyoxalerythropoietin were as effectiveas native erythropoietin for neural cells in vitro as shown in FIGS.4-6. In-vitro testing was carried out using neural-like embryonalcarcinoma cells (P19) that undergo apoptosis upon the withdrawal ofserum. Twenty-four hours before the removal of serum, 1-1000 ng/ml oferythropoietin or a modified erythropoietin was added to the cultures.The following day the medium was removed, the cells washed with fresh,non-serum containing medium, and medium containing the test substance(no serum) added back to the cultures for and additional 48 hours. Todetermine the number of viable cells, a tetrazolium reduction assay wasperformed (CellTiter 96; Promega, Inc.). As FIG. 4-5 illustrate,asialoerythropoietin appears to be of equal potency to erythropoietinitself in preventing cell death. The phenylglyoxal-modifiederythropoietin was tested using the neural-like P19 cell assay describedabove. As FIG. 6 illustrates, this chemically-modified erythropoietinfully retains its neuroprotective effects.

Retention of neuroprotective activity in vivo was confirmed using a ratfocal ischemia model in which a reversible lesion in the territory ofthe middle cerebral artery is performed as described previously (Brineset al., 2000, Proc. Nat. Acad. Sci. U.S.A. 97:10526-31). Adult maleSprague-Dawley rats were administered asialoerythropoietin orerythropoietin (5000 U/kgBW intraperitoneally) or vehicle at the onsetof the arterial occlusion. Twenty-four hours later, the animals weresacrificed and their brains removed for study. Serial sections were cutand stained with tetrazolium salts to identify living regions of thebrain. As shown in FIG. 7, asialoerythropoietin was as effective asnative erythropoietin in providing neuroprotection from 1 hour ofischemia. FIG. 8 shows the results of another focal ischemia model inwhich a comparative dose response was performed with erythropoietin andasialoerythropoietin. At the lowest dose of 250 U/kg,asialoerythropoietin afforded protection whereas unmodifiederythropoietin did not.

Example 5 Modification of Primary Structure of Erythropoietin andEffectiveness at Neuronal Protection

A number of mutant erythropoietin molecules have been described which donot bind to the erythrocyte erythropoietin receptor and thus do notsupport erythropoiesis in vivo or in vitro. Some of these molecules willnevertheless mimic the actions of erythropoietin itself in other tissuesor organs. For example, a 17-mer containing the amino-acid sequence of31-47 of native erythropoietin is inactive for erythropoiesis but fullyactive for neural cells in vitro (Campana & O'Brien, 1998: Int. J. Mol.Med. 1:235-41).

Derivative erythropoietin desirable for the uses described herein may begenerated by guanidination, amidination, trinitrophenylation,acetylation, succinylation, nitration, or modification of arginineresidues or carboxyl groups, among other procedures as mentioned hereinabove, to produce erythropoietins which maintain their activities forspecific organs and tissues but not for others, such as erythrocytes.When erythropoietin is subjected to the above reactions, it has beenfound that in general the resultant molecule lacks both in-vivo andin-vitro erythropoietic activity (e.g., Satake et al; 1990, Biochim.Biophys. Acta 1038:125-9). Some examples of the preparation of modifiederythropoietins are described below.

Biotinylation at free amino groups of erythropoietin. 0.2 mgD-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester (BoehringerMannheim #1418165) was dissolved in 100 ul DMSO. This solution wascombined with 400 ul PBS containing approximately 0.2 mg erythropoietinin a foil covered tube. After incubation for 4 hours at roomtemperature, the unreacted biotin was separated by gel filtration on aCentricon 10 column. As shown by FIG. 10, this biotinylatederythropoietin protects p19 cells from serum withdrawal.

In “Biotinylated recombinant human erythropoietins: Bioactivity andUtility as a receptor ligand” by Wojchowski et al. Blood, 1989,74(3):952-8, the authors use three different methods of biotinylatingerythropoietin. Biotin is added to (1) the sialic acid moieties (2)carboxylate groups (3) amino groups. The authors use a mouse spleen cellproliferation assay to demonstrate that (1) the addition of biotin tothe sialic acid moieties does not inactivate the biological activity oferythropoietin (2) the addition of biotin to carboxylate groups led tosubstantial biological inactivation of erythropoietin (3) the additionof biotin to amino groups resulted in complete biological inactivationof erythropoietin. These methods and modifications are fully embracedherein. FIG. 9 shows the activity of biotinylated erythropoietin andasialoerythropoietin in the serum-starved P19 assay.

Iodination of erythropoietin. Method 1—Iodo Beads. One Iodo Bead(Pierce, Rockford, Ill.) was incubated in 100 ul PBS (20 mM sodiumphosphate, 0.15M NaCl, pH7.5) containing 1 mCi free Na¹²⁵I for 5minutes. 100 ug erythropoietin in 100 ul PBS was then added to themixture. After a ten minute incubation period at room temperature, thereaction was stopped by removing the 200 ul solution from the reactionvessel (leaving the iodo bead behind). The excess iodine was removed bygel filtration on a Centricon 10 column. As shown in FIG. 11,iodo-erythropoietin produced in this manner is efficacious in protectingP19 cells from serum withdrawal.

Method 2 —Chloramine T. 100 ug erythropoietin in 100 ul PBS was added to500 uCi Na¹²⁵I were mixed together in an eppendorf tube. 25 ulchloramines T (2 mg/ml) was then added and the mixture was incubated for1 minute at room temperature. 50 ul of Chloramine T stop buffer (2.4mg/ml sodium metabisulfite, 10 mg/ml tyrosine, 10% glycerol, 0.1% xylenein PBS was then added. The iodotyrosine and iodinated erythropoietinwere then separated by gel filtration on a Centricon 10 column.

Lysine modifications: Carbamylation: erythropoietin (100 ug) wasmodified with potassium cyanate as described in Plapp et al (“Activityof bovine pancreatic deoxyribonuclease A with modified amino groups”1971, J. Biol. Chem. 246, 939-845).

Trinitrophenylation: erythropoietin (100 ug) was modified with2,4,6-trinitrobenzenesulfonate as described in Plapp et at (“Activity ofbovine pancreatic deoxyribonuclease A with modified amino groups” 1971,J. Biol. Chem. 246, 939-845)

Acetylation: erythropoietin (100 ug) was incubated in 0.3M phosphatebuffer (pH7.2) containing an equal amount of acetic anhydride at 0 C for1 hour. The reaction was stopped by dialysis against distilled water.

Succinylation: erythropoietin (100 ug) in 0.5 M NaHCO₃ (pH 8.0) wasincubated with a 15 molar excess of succinic anhydride at 15 C for 1hour. The reaction was stopped by dialysis against distilled water.

Arginine modifications: erythropoietin was modified with 2,3 butanedioneas described in Riordan (“Functional arginyl residues incarboxypeptidase A. Modification with butanedione” Riordan J F,Biochemistry 1973, 12(20): 3915-3923).

Erythropoietin was modified with cylcohexanone as in Patthy et al(“Identification of functional arginine residues in ribonuclease A andlysozyme” Patthy, L, Smith E L, J. Biol. Chem. 1975 250(2): 565-9).

Erythropoietin was modified with phenylglyoxal as described in Werber etal. (“Proceedings: Carboxypeptidase B: modification of functionalarginyl residues” Werber, M M, Sokolovsky M Isr J Med Sci 1975 11(11):1169-70).

Tyrosine modifications: erythropoietin (100 ug) was incubated withtetranitromethane as previously described in Nestler et al “Stimulationof rat ovarian cell steroidogenesis by high density lipoproteinsmodified with tetranitromethane” Nestler J E, Chacko G K, Strauss J F3rd. J Biol Chem 1985 Jun. 25; 260(12):7316-21).

Glutamic acid (and aspartic acid) modifications: In order to modifycarboxyl groups, erythropoietin (100 ug) was incubated with 0.02 M EDCin 1M glycinamide at pH 4.5 at room temperature for 60 minutes asdescribed in Carraway et al “Carboxyl group modification in chymotrypsinand chymotrypsinogen.” Carraway K L, Spoerl P, Koshland D E Jr. J MolBiol 1969 May 28; 42(1):133-7.

Tryptophan residue modifications: erythropoietin (100 ug) was incubatedwith 20 uM n-bromosuccinimide in 20 mM potassium phosphate buffer (pH6.5) at room temperature as described in Ali et al., J Biol Chem. 1995Mar. 3; 270(9):4570-4. The number of oxidized tryptophan residues wasdetermined by the method described in Korotchkina (Korotchkina; L G etal Protein Expr Purif. 1995 February; 6(1):79-90).

Removal of amino groups: In order to remove amino groups oferythropoietin (100 ug) was incubated with in PBS (pH 7.4) containing 20mM ninhydrin (Pierce Chemical, Rockford, Ii), at 37 C for two hours asin Kokkini et al (Kokkini, G., et al “Modification of hemoglobin byninhydrin” Blood, Vol. 556, No 4 1980: 701-705). Reduction of theresulting aldehyde was accomplished by reacting the product with Sodiumborohydride or lithium aluminum hydride. Specifically, erythropoietin(100 ug) was incubated with 0.1M sodium borohydride in PBS for 30minutes at room temperature. The reduction was terminated by cooling thesamples on ice for 10 minutes and dialyzing it against PBS, three times,overnight. (Kokkini, G., Blood, Vol. 556, No 4 1980: 701-705). Reductionusing lithium aluminum hydride was accomplished by incubatingerythropoietin (100 ug) with 0.1M lithium aluminum hydride in PBS for 30minutes at room temperature. The reduction was terminated by cooling thesamples on ice for 10 minutes and dialyzing it against PBS, three times,overnight.

Disulfide reduction and stabilization: erythropoietin (100 ug) wasincubated with 500 mM DTT for 15 minutes at 60 C. 20 mM iodoacetamide inwater was then added to the mixture and incubated for 25 minutes, atroom temperature in the dark.

Limited proteolysis: Erythropoietin can be subjected to a limitedchemical proteolysis that targets specific residues. Erythropoietin wasreacted with 2-(2-nitrophenylsulfenyl)-3-methyl-3% bromoindolenine whichcleaves specifically after tryptophan residues in a 50 times excess in50% acetic acid for 48 hours in the dark at room temperature in tubescapped under nitrogen pressure. The reaction was terminated by quenchingwith tryptophan and desalting.

Example 6 Protection of Retinal Ischemia by Peripherally-AdministeredErythropoietin

Retinal cells are very sensitive to ischemia such that many will dieafter 30 minutes of ischemic stress. Further, subacute or chronicischemia underlies the deterioration of vision which accompanies anumber of common human diseases, such as diabetes mellitus, glaucoma,and macular degeneration. At the present time there are no effectivetherapies to protect cells from ischemia. A tight endothelial barrierexists between the blood and the retina that excludes most largemolecules. To test whether peripherally-administered erythropoietin willprotect cells sensitive to ischemia, an acute, reversible glaucoma ratmodel was utilized as described by Rosenbaum et al. (1997; Vis. Res.37:3443-51). Specifically, saline was injected into the anterior chamberof the eye of adult male rats to a pressure above systemic arterialpressure and maintained for 60 minutes. Animals were administered salineor 5000 U erythropoietin/kg body weight intraperitoneally 24 hoursbefore the induction of ischemia, and continued as a daily dose for 3additional days. Electroretinography was performed on dark-adapted rats1 week after treatment. FIG. 11-12 illustrate that the administration oferythropoietin is associated with to good preservation of theelectroretinogram (ERG) (Panel D), in contrast to animals treated withsaline alone (Panel C), for which very little function remained. FIG. 11compares the electroretinogram a- and b-wave amplitudes for theerythropoietin-treated and saline-treated groups, and shows significantprotection afforded by erythropoietin.

Example 7 Restorative Effects of Erythropoietin on Diminished CognitiveFunction Arising from Brain Injury

In a study to demonstrate the ability of erythropoietin to restorediminished cognitive function in mice after receiving brain trauma,female Balb/c mice were subject to blunt brain trauma as described inBrines et al. PNAS 2000, 97; 10295-10672 and five days later, dailyerythropoietin administration of 5000 U/kg-bw intraperitoneally wasbegun. Twelve days after injury, animals were tested for cognitivefunction in the Morris water maze, with four trials per day. While bothtreated and untreated animals performed poorly in the test (with swimtimes of about 80 seconds out of a possible 90 seconds), FIG. 13 showsthat the erythropoietin-treated animals performed better (in thispresentation, a negative value is better). Even if the initiation oferythropoietin treatment is delayed until 30 days after trauma (FIG.14), restoration of cognitive function is also seen.

Example 8 Kainate Model

In the kainate neurotoxicity model, asialoerythropoietin wasadministered according to the protocol of Brines et al. Proc. Nat. Acad.Sci. U.S.A. 2000, 97; 10295-10672 at a dose of 5000 U/kg-bw givenintraperitoneally 24 hours before the administration of 25 mg/kg kainateis shown to be as effective as erythropoietin, as shown by time to death(FIG. 15).

The invention is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the invention, and functionally equivalent methods andcomponents are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference herein intheir entireties for all purposes.

1.-57. (canceled)
 58. A method for protecting, maintaining, enhancing or restoring the function or viability of an erythropoietin-responsive mammalian cell, or its associated cells, tissues, or organs, comprising administering to a mammal a pharmaceutical composition comprising a therapeutically effective amount of an erythropoietin having at least one of the following modifications: i) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acid moieties; ii) a reduced number or no N-linked carbohydrates; iii) one or more modified lysine residues; or iv) a modification of the N-terminal amino group of the erythropoietin molecule, wherein the modified erythropoietin has (a) tissue protective activity as determined in vitro by the P19 assay or in vivo by the middle cerebral artery lesion assay and (b) a reduced level of erythropoietic activity compared to native erythropoietin, and wherein said mammalian cell or its associated cells, tissues, or organs is central nervous system or peripheral nervous system cells, tissues, or organs.
 59. The method of claim 58 wherein a lysine residue of said modified erythropoietin is carbamylated.
 60. The method of claim 58 wherein said modified erythropoietin is asialoerythropoietin.
 61. The method of claim 58 wherein said modified erythropoietin is non-erythropoietic.
 62. The method of claim 58 wherein said modified erythropoietin is effective for the maintenance, restoration, or enhancement of cognitive function in a mammal having or at risk for cognitive dysfunction.
 63. The method of claim 62 wherein said cognitive dysfunction is caused by brain trauma.
 64. The method of claim 62 wherein said enhancement of cognitive function is enhancement of learning.
 65. The method of claim 58 wherein the mammal has, has had, is at risk for, or is undergoing, a seizure disorder, multiple sclerosis, stroke, central nervous system injury, neuronal loss, ischemia, cerebral ischemia, focal ischemia, subarachnoid bleeds, aneurysm, aneurismal bleeds, inflammation, age-related loss of cognitive function, neurodegenerative disease or disorder, Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, Leigh disease, Guillain Barre, dementia, AIDS dementia, senile dementia, Lewy body dementia, memory loss, amyotrophic lateral sclerosis, alcoholism, neuropsychiatric or neuropsychologic disorder, mood disorder, uni-polar affective disorder, depression, major depressive disorder, dysthymic disorder, mania, bipolar affective disorder, anxiety disorder, anxiety, schizophrenia, schizoaffective disorder, obsessive-compulsive disorder, attention deficit disorder, attention-deficit hyperactivity disorder, autism, a prion disease such as Creutzfeldt-Jakob disease, Friedreich's ataxia, Wilson's disease, trauma, brain trauma, blunt trauma, concussive injury, brain or spinal cord injury, post-operative cognitive dysfunction, post-operative treatment for embolic or ischemic injury, diabetic neuropathy, loss of cognitive function, neurotoxicity, subdural hematoma, muscular dystrophy, myotonic dystrophy, or panic disorder.
 66. The method of claim 58 wherein the pharmaceutical composition is formulated for administration before the onset of an injury, disease, or condition.
 67. A method for protecting, maintaining, enhancing or restoring the function or viability of an erythropoietin-responsive mammalian cell, or its associated cells, tissues, or organs, comprising administering to a mammal a pharmaceutical composition comprising a therapeutically effective amount of an erythropoietin having at least one of the following modifications: i) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acid moieties; ii) a reduced number or no N-linked carbohydrates; iii) one or more modified lysine residues; or iv) a modification of the N-terminal amino group of the erythropoietin molecule, wherein the modified erythropoietin has (a) tissue protective activity as determined in vitro by the P19 assay or in vivo by the middle cerebral artery lesion assay and (b) a reduced level of erythropoietic activity compared to native erythropoietin, and wherein said mammalian cell or its associated cells, tissues, or organs is heart, myocardium, or coronary artery.
 68. The method of claim 67 wherein a lysine residue of said modified erythropoietin is carbamylated.
 69. The method of claim 67 wherein said modified erythropoietin is asialoerythropoietin.
 70. The method of claim 67 wherein said modified erythropoietin is non-erythropoietic.
 71. The method of claim 67 wherein the mammal has, has had, is at risk for, or is undergoing, cardiac arrest, myocardial infarction, heart-lung bypass, heart injury, myocardium injury, heart trauma, chronic heart failure, or coronary artery occlusion.
 72. The method of claim 67 wherein the pharmaceutical composition is formulated for administration before the onset of an injury, disease, or condition.
 73. A method for protecting, maintaining, enhancing or restoring the function or viability of an erythropoietin-responsive mammalian cell, or its associated cells, tissues, or organs, comprising administering to a mammal a pharmaceutical composition comprising a therapeutically effective amount of an erythropoietin having at least one of the following modifications: i) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acid moieties; ii) a reduced number or no N-linked carbohydrates; iii) one or more modified lysine residues; or iv) a modification of the N-terminal amino group of the erythropoietin molecule, wherein the modified erythropoietin has (a) tissue protective activity as determined in vitro by the P19 assay or in vivo by the middle cerebral artery lesion assay and (b) a reduced level of erythropoietic activity compared to native erythropoietin, and wherein said mammalian cell or its associated cells, tissues, or organs is eye, retina, or kidney.
 74. The method of claim 73 wherein a lysine residue of said modified erythropoietin is carbamylated.
 75. The method of claim 73 wherein said modified erythropoietin is asialoerythropoietin.
 76. The method of claim 73 wherein said modified erythropoietin is non-erythropoietic.
 77. The method of claim 73 wherein the mammal has, has had, is at risk for, or is undergoing, eye tissue damage, macular degeneration, diabetic retinopathy, glaucoma, retinal ischemia, retinal trauma, retinitis pigmentosa, optic nerve damage, retinal detachment, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery blockage, retinal vein blockage, kidney injury, renal failure, ischemic renal failure, diabetic kidney disease, or a nephrotic syndrome.
 78. The method of claim 73 wherein the pharmaceutical composition is formulated for administration before the onset of an injury, disease, or condition. 